Device and method for regeneration and repair of cartilage lesions

ABSTRACT

Disclosed is a cartilage repair product that induces both cell ingrowth into a bioresorbable material and cell differentiation into cartilage tissue. Such a product is useful for regenerating and/or repairing both vascular and avascular cartilage lesions, particularly articular cartilage lesions, and even more particularly mensical tissue lesions, including tears as well as segmental defects. Also disclosed is a method of regenerating and repairing cartilage lesions using such a product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part under 35 U.S.C. § 120 of PCTApplication No. PCT/EP 98/05100, entitled “COMPOSITION AND DEVICE FOR INVIVO CARTILAGE REPAIR COMPRISING NANOCAPSULES WITH OSTEOINDUCTIVE AND/ORCHONDROINDUCTIVE FACTORS”, filed Aug. 12, 1998, which designates theUnited States and which claims priority from European Application No. EP97810567.4, entitled “COMPOSITION AND DEVICE FOR IN VIVO CARTILAGEREPAIR”, filed Aug. 14, 1997. The entire disclosures of Application Nos.PCT/EP 98/05100 and EP 97810567.4 are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a cartilage regeneration and repairproduct that induces cell ingrowth into a bioresorbable material andcell differentiation into cartilage tissue, and to methods of using sucha product to repair cartilage lesions.

BACKGROUND OF THE INVENTION

Articular cartilage, an avascular tissue found at the ends ofarticulating bones, has limited natural capacity to heal. During normalcartilage ontogeny, mesenchymal stem cells condense to form areas ofhigh density and proceed through a series of developmental stages thatends in the mature chondrocyte. The final hyaline cartilage tissuecontains only chondrocytes that are surrounded by a matrix composed oftype II collagen, sulfated proteoglycans, and additional proteins. Thematrix is heterogenous in structure and consists of threemorphologically distinct zones: superficial, intermediate, and deep.Zones differ among collagen and proteoglycan distribution,calcification, orientation of collagen fibrils, and the positioning andalignment of chondrocytes (Archer et al., 1996, J. Anat. 189(1):23-35;Morrison et al., 1996, J. Anat. 189(1): 9-22; and Mow et al.,1992,Biomaterials 13(2): 67-97). These properties provide the uniquemechanical and physical parameters to hyaline cartilage tissue.

The meniscus, a C-shaped cartilaginous tissue, performs severalfunctions in the knee including load transmission from the femur to thetibia, stabilization in the anterior-posterior position during flexion,and joint lubrication. Damage to the meniscus results in reduced kneestability and knee locking. Over 20 years ago, meniscectomies wereperformed which permitted immediate pain relief, but were subsequentlyfound to induce the early onset of osteoarthritis (Fairbank, J. BoneJoint Surg. 30B: 664-670; Allen et al., 1984, J. Bone Joint Surg.66B:666-671; and Roos et al., 1998, Arth. Rheum. 41:687-693). Morerecently, partial meniscectomies and repair of meniscal tears have beenperformed (FIGS. 9A-D; Jackson, D., ed., 1995, Reconstructive KneeSurgery Master Techniques in Orthopedic Surgery, ed. R. Thompson, RavenPress: New York). However, partial resection results in the loss offunctional meniscus tissue and the early onset of osteoarthritis (Lynchet al., 1983, Clin. Orthop. 172:148-153; Cox et al., 1975, Clin. Orthop.109:178-183; King, 1995, J. Bone Joint Surg. 77B:836-837). Additionally,repair of meniscal tears is limited to tears in the vascular ⅓ of themeniscus; tears in the semivascular to avascular ⅔ are not repairable(FIGS. 9A-D; Jackson, ibid.). Of the approximately, 560,000 meniscalinjuries that occur annually in the United States, an estimated 80% oftears are located in the avascular, irreparable zone. Clearly, a methodthat both repairs “non-repairable” tears or that can induce regenerationof resected menisci would be valuable for painless musculoskeletalmovement and prevention of the early onset of osteoarthritis in a largesegment of the population.

The proximal, concave surface of the meniscus contacts the femoralcondyle and the distal, flat surface contacts the tibial plateaus. Theouter one-third of the meniscus is highly vascularized and containsdense, enervated, connective tissue. In contrast, the remaining meniscusis semivascular or avascular, aneural tissue consisting offibrochondrocytes surrounded by abundant extracellular matrix (McDevittet al., Clin. Orthop. Rel. Res. 252:8-17). Fibrochondrocytes aredistinctive in both appearance and function compared to undifferentiatedfibroblasts. Fibroblasts are elongated cells containing many cellularprocesses and produce predominantly type I collagen. The matrix producedby fibroblasts does not produce a sufficient mechanical load. Incontrast, fibrochondrocytes are round, and are encompassed by lacunaethat consists of type I and type II collagen and proteoglycans. Thesematrix components support compressive forces that are commonly exertedon the meniscus during musculoskeletal movement.

In the 1960's, demineralized bone matrix was observed to induce theformation of new cartilage and bone when implanted in ectopic sites(Urist, 1965, Science 150:893-899). The components responsible for theosteoinductive activities were termed Bone Morphogenetic Proteins (BMP).At least seven individual BMP proteins were subsequently identified frombone (BMP 1-7) and amino acid analysis revealed that six of the sevenBMPs were related to each other and to other members of the TGF-βsuperfamily. During endochondral bone formation, TGF-β family membersdirect a cascade of events that includes chemotaxis, differentiation ofpluripotential cells to the cartilage lineage, maturation ofchondrocytes to the hypertrophic stage, mineralization of cartilage,replacement of cartilage with bone cells, and the formation of acalcified matrix (Reddi, 1997, Cytokine & Growth Factor Reviews8:11-20). Although individual, recombinant BMPs can induce these events,the prevalence of multiple TGF-β family members in bone tissue underliesthe complexity involved in natural osteogenesis.

Bone Protein (Sulzer Orthopedics Biologics, Denver, Colo.), alsoreferred to herein as BP, is a naturally derived mixture of proteinsisolated from demineralized bovine bones that has osteogenic activity invitro and in vivo. In the rodent ectopic model, BP induces endochondralbone formation or bone formation through a cartilage intermediate(Damien, C. et al., 1990, J. Biomed. Mater. Res. 24:639-654). BP incombination with calcium carbonate promotes bone formation in the body(Poser and Benedict, PCT Publication No. WO95/13767). In vitro, BP hasbeen shown to promote differentiation to cartilage of murine embryonicmesenchymal stem cells (Atkinson et al., 1996, In “Molecular andDevelopmental Biology of Cartilage”, Bethesda, Md., Annals New YorkAcad. Sci. 785:206-208; Atkinson et al., 1997, J. Cell. Biochem.65:325-339) and of adult myoblast and dermal cells (Atkinson et al.,1998, 44th Annual Meeting, Orthopaedic Research Society, abstract). Toensure chondrogenesis in these in vitro systems, however, cultureconditions must be tightly controlled throughout the culture period,including by controlling cellular organization within the culture,optimizing media formulations, and adding exogenous factors that must becarefully established to maximize chondrogenesis over mitogenesis. Suchoptimization of conditions makes the application of the disclosed invitro methods to an in vivo system unrealistic and unpredictable. Inaddition, although in vitro cultures of adult myoblast and dermal cellsinitially resulted in chondrogenesis, the effect was only transient andover time, the cultures reverted to their original phenotype. Althoughcertain embryonic and precursor cell types showed prolongedchrondrogenesis in vitro in these studies, it would be unpredictable oreven impossible in the case of embryonic cells that these specific celltypes could be recruited to a site in vivo in an adult patient.

Atkinson et al., in PCT Application No. PCT/EP/05100, incorporatedherein by reference in its entirety, describe a delivery system forosteoinductive and/or chondroinductive mixture of naturally derivedfactors for the induction of cartilage repair.

Hunziker (U.S. Pat. Nos. 5,368,858 and 5,206,023) describes a cartilagerepair composition consisting of a biodegradable matrix, a proliferationand/or chemotactic agent, and a transforming factor. A two-stageapproach is used where each component has a specific function over time.First, a specific concentration of proliferation/chemotactic agent fillsthe defect with repair cells. Second, a larger transforming factorconcentration, preferably provided in conjunction with a deliverysystem, transforms repair cells to chondrocytes. The second stagedelivery of a high concentration of transforming factor in a deliverysystem (i.e., liposomes) was required to obtain formation of hyalinecartilage tissue at the treatment site.

Chen and Jeffries (U.S. Pat. No. 5,707,962) describe osteogeniccompositions consisting of collagen and sorbed factors to enhanceosteogenesis.

Valee and King (U.S. Pat. No. 4,952,404) describe healing of injured,avascular meniscus tissue by release of the angiogenic factor,angiogenin, over at least 3 weeks.

Previously, Amoczky et al. described a method using an autogenous fibrinclot to repair an avascular, circular lesion in canine menisci (Amoczkyet al., 1988, J. Bone Joint Surg. 70A:1209-1217). This approach enhancedrepair of meniscal tissue compared to controls lacking the fibrin clot.However, the repair tissue was not meniscus-like tissue, but ratherconnective scar tissue.

Hashimoto et al. described a method using fibrin sealant with or withoutendothelial cell growth factor in avascular, circular meniscal defectsin the canine model (Hashimoto et al., 1992, Am. J. Sports Med.20:537-541). The growth factor added a modest benefit compared tohealing with fibrin sealant alone and this additional effect was notobserved until three months after treatment, indicating an indirectcontribution of the growth factor. In addition, the defect was filledwith hyaline cartilage-like cells, which are not typically present innormal meniscus tissue.

Shirakura, et al. describe the use of an autogenous synovium graftsutured into meniscal tears. While the synovium did enhance healing in ⅓of the animals, the grafts healed with fibrous tissue, notfibrocartilaginous tissue normally observed in meniscus tissue(Shirakura, 1997, Acta. Orthop. Scand. 68:51-54). Furthermore, ⅔ of thegrafts did not heal.

The molecular mechanism for cartilage and bone formation has beenpartially elucidated. Both bone morphogenetic proteins (BMP) andtransforming growth factor β (TGFβ) molecules bind to cell surfacereceptors (i.e., TGFβ/BMP receptors) to initiate a cascade of signals tothe nucleus that promotes proliferation, differentiation to cartilage,and/or differentiation to bone (Massague, 1996, Cell 85:947-950). In1984, Urist described a substantially pure, but not recombinant, BMPcombined with a biodegradable poly(lactic acid)polymer delivery systemfor bone repair (U.S. Pat. No. 4,563,489). This system blends togetherequal quantities of BMP and poly(lactic acid) (PLA) powder (100 μg ofeach) and decreases the amount of BMP required to promote bone repair.

Hattersley et al. (WO 96/39170) disclose a two factor composition forinducing cartilaginous tissue formation using a cartilageformation-inducing protein and a cartilage maintenance-inducing protein.Specific recombinant cartilage inducing proteins are specified asBMP-13, MP-52 and BMP-12, and specific cartilage maintenance-inducingproteins are specified as BMP-9. In one embodiment, BMP-9 isencapsulated in a resorbable polymer system and delivered to coincidewith the presence of cartilage formation inducing protein(s).

Laurencin et al. (U.S. Pat. No. 5,629,009) disclose achondrogenesis-inducing device, consisting of a polyanhydride andpolyorthoester, that delivers water soluble proteins derived fromdemineralized bone matrix, TGFβ, epidermal growth factor (EGF),fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF).

Previously, Li and Stone (U.S. Pat. No. 5,681,353) have described aMeniscal Augmentation Device that consists of biocompatible andbioresorbable fibers that acts as a scaffold for the ingrowth ofmeniscal fibrochondrocytes, supports normal meniscal loads, and has anouter surface that approximates the natural meniscus contour. Afterpartial resection of the meniscus to the vascular zone, this device isimplanted into the resulting segmental defect. The results have beendescribed in both canines and humans (Stone et al., 1992, Am. J. SportsMed. 20:104-111; and Stone et al., 1997, J. Bone Joint Surg.79:17701777).

The Meniscus Augmentation Device, the research reports described above,and current repair surgeries provide encouraging results in the area ofcartilage repair, but are not satisfactory to induce repair of“non-repairable” avascular tears in which the repair tissue is meniscustissue, and are not satisfactory to produce short patient rehabilitationtimes and regenerated meniscus tissue in the vascular zone. Furthermore,no reports have been described in which demonstrate enhanced healingrates of “repairable” meniscal tears in vivo.

SUMMARY OF THE INVENTION

The present invention relates to a product and method for repairingand/or regenerating cartilage lesions. The product and method of thepresent invention are useful for repairing a variety of cartilagelesions, including articular and mensical lesions, including vascular,semivascular and avascular lesions. Moreover, the product and method ofthe present invention can be used to repair different sizes and shapesof cartilage lesions, including radial tears, bucket handle tears, andsegmental defects.

One embodiment of the present invention is directed to a product forrepair of cartilage lesions. The product includes: (a) a cartilagerepair matrix; and (b) a cartilage-inducing composition associated withthe matrix for provision of chondrogenesis-enhancing proteins. Each ofthe chondrogenesis-enhancing proteins can be provided as a proteinand/or by a recombinant nucleic acid molecule encoding the protein, suchrecombinant nucleic acid molecule being operatively linked to atranscription control sequence, and mixtures of proteins and recombinantnucleic acid molecules can be provided. The mixture ofchondrogenesis-enhancing proteins include at least a first and secondtransforming growth factor β (TGFβ) superfamily protein, wherein thefirst and second TGFβ superfamily protein are different, and at leastone protein from a family of proteins selected from growth factorproteins and bone matrix proteins. The chondrogenesis-enhancing proteinsare further characterized in that, when cultured together with ATDC5cells for seven days at a concentration of about 100 ng/ml or less,induce a statistically significant increase in A₅₉₅ in an Alcian Blueassay performed with said cells.

The product of the present invention can also be formulated to include:(a) a cartilage repair matrix; and (b) a cartilage-inducing compositionassociated with the matrix, which includes cells that have been culturedwith the above-described mixture of chondrogenesis-enhancing proteins.

The cartilage repair matrix of a shape and size that conforms to thecartilage defect such that the defect is repaired. As such, the matrixcan be configured as a sheet, which is most suitable for repairingcartilage tears, or the matrix can be configured to repair a segmentaldefect, which can include a tapered shape. The cartilage repair matrixcan be formed of any suitable material, including synthetic polymericmaterial and ground substances. In one embodiment, the matrix isbioresorbable. In another embodiment, the matrix is porous. When thematrix is configured as a sheet, the matrix is preferably notcross-linked, and when the matrix is configured to repair a segmentaldefect, the matrix is preferably cross-linked.

The cartilage-inducing composition can be associated with the matrix byany suitable method, including, but not limited to freeze-drying thecomposition onto a surface of said matrix and suspension within saidcartilage repair matrix of a delivery formulation containing saidcomposition. Additionally, the composition can be associated with thematrix ex vivo or in vivo. In one embodiment, a suitable deliveryformulation includes nanospheres, wherein the nanospheres are polymerparticles having a size of less than 1000 nm and being loaded withbetween 0.001% and 17% by weight of the cartilage-inducing composition.The nanospheres have an in vitro analytically determined release rateprofile with an initial burst of about 10% to about 20% of the totalamount of the composition over a first 24 hour period and a long timerelease rate of a least 0.1% per day during at least seven followingdays. Yet another suitable delivery vehicle includes a liposome. Whenthe chondrogenesis-enhancing proteins are provided as recombinantnucleic acid molecule, the recombinant nucleic acid molecule can beprovided in any suitable form for delivery, including as naked DNA,transformed into a recombinant cell or provided in the form of arecombinant virus.

In preferred embodiments, the TGFβ superfamily proteins included in thechondrogenesis-enhancing protein mixture include, but are not limitedto, TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein (BMP)-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derivedmorphogenetic protein (CDMP)-1, CDMP-2, and/or CDMP-3; and morepreferably include TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein(BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, and/or a cartilage-derivedmorphogenetic protein (CDMP). The bone matrix proteins included in thechondrogenesis-enhancing protein mixture include, but are not limitedto, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase,cathepsin L pre, osteopontin, matrix GLA protein (MGP), biglycan,decorin, proteoglycan chondroitin sulfate-III (PG-CS III), bone acidicglycoprotein-75 (BAG-75), thrombospondin (TSP) and fibronectin; and morepreferably include osteocalcin, osteonectin, MGP, BSP, lysyloxidase andcathepsin L pre. The growth factor proteins included in thechondrogenesis-enhancing protein mixture include, but are not limitedto, fibroblast growth factor I (FGF-I), FGF-II, FGF-9, leukocyteinhibitory factor (LIF), insulin, insulin growth factor I (IGF-I),IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB,stromal derived factor-2 (SDF-2), pituitary thyroid hormone (PTH),growth hormone, hepatocyte growth factor (HGF), epithelial growth factor(EGF), transforming growth factor-α (TGFα) and hedgehog proteins; andmore preferably include at least fibroblast growth factor I (FGF-I). Thechondrogenesis-enhancing proteins can also include one or more serumproteins, including, but not limited to albumin, transferrin, α2-HsGlycoP, IgG, α1-antitrypsin, β2-microglobulin, Apo A1 lipoprotein (LP)and Factor XIIIb; and more preferably include albumin, transferrin, ApoA1 LP and Factor XIIIb. Particularly preferred mixtures ofchondrogenesis-enhancing proteins include TGFβ1, TGFβ2, TGFβ3, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin,osteonectin, MGP, BSP, lysyloxidase, and cathepsin L pre, and in anotherembodiment, TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6,BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase,cathepsin L pre, albumin, transferrin, Apo A1 LP and Factor XIIIb. Yetanother preferred mixture of chondrogenesis-enhancing proteins are boneprotein (BP).

Another embodiment of the present invention relates to a method forrepair of cartilage lesions, which includes the steps of implanting andfixing into a cartilage lesion a cartilage repair product of the presentinvention, as described above. The method of the present invention canbe used to enhance the rate and/or quality of repair of vascularcartilage tears and segmental defects, and can provide the ability torepair semivascular and avascular tears and segmental defects that,prior to the present invention, were typically considered to beirreparable. When the lesion is in semivascular or avascular cartilage,the product can additionally include a time controlled deliveryformulation.

In one aspect, the method of the present invention includes the use oftwo cartilage repair products to repair a segmental defect. The firstproduct includes a cartilage repair matrix, which is configured as asheet, is associated with the chondrogenesis-enhancing proteins asdescribed above. The second product includes a cartilage repair matrixconfigured to replace cartilage removed from the segmental defect, whichmay or may not be associated with the chondrogenesis-enhancing proteinsof the present invention.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIG. 1A shows a meniscal radial tear.

FIG. 1B shows a conventional suture repair and resection of the meniscalradial tear illustrated in FIG. 1A.

FIG. 1C shows a meniscal triple bucket handle tear.

FIG. 1D shows a conventional suture repair and resection of the meniscaltriple bucket handle tear illustrated in FIG. 1C.

FIG. 2A illustrates implantation of a cartilage repair product of thepresent invention to a meniscal segmental lesion.

FIG. 2B illustrates fixation of a cartilage repair product of thepresent invention to a meniscal segmental lesion.

FIG. 3A is a diagram illustrating a meniscus cross section havingvascular, semi-vascular and avascular zones.

FIG. 3B illustrates one approximate shape of a cartilage repair productof the present invention.

FIG. 4A is an illustration of a meniscus having a longitudinal tear inthe avascular region as viewed from the femur towards the tibia.

FIG. 4B is a diagram illustrating a cross section of the meniscusdepicted in FIG. 4A containing a cartilage repair product of the presentinvention.

FIG. 5 is a line graph showing quantitation of Alcian Blue staining ofATDC5 micromass cultures.

FIG. 6 is a bar graph showing quantitation of Alcian Blue staining ofATDC5 micromass cultures in Nutridoma-containing media at 7 and 14 days.

FIG. 7 is a bar graph showing quantitation of Alcian Blue staining ofATDC5 micromass cultures containing HPLC fractions of proteins isolatedfrom demineralized bovine bones.

FIG. 8 is a diagram illustrating a cross-section view of a combinationcollagen meniscus implant (CMI) and sheet cartilage repair product ofthe present invention used to repair a meniscal defect.

DETAILED DESCRIPTION OF THE INVENTION

The present application generally relates to a product for repairingand/or regenerating cartilage lesions, and methods of repairing orregenerating cartilage lesions using such a product. The product andmethods of the present invention are particularly useful for repairingdefects (i.e., lesions) in articular cartilage (e.g., hyaline cartilage)and meniscal cartilage (e.g., fibrocartilage). When used to repairmeniscal cartilage, the product and method of the present invention areeffective for repairing both vascular and avascular meniscal cartilagelesions. In particular, the product and method of the present inventionincrease the rate of meniscus repair and induce more normal (i.e.,endogenous-type) meniscal tissue than is commonly observed during theconventional repair practiced currently. The cartilage repair product ofthe present invention can also induce meniscus repair of avascular,“irreparable” tears and, furthermore, fill the defect with meniscus-liketissue. Moreover, the product and method are useful for repairing andregenerating meniscal tissue which has been removed by partial orcomplete meniscectomy. The product and method of the present inventioncan enhance blood vessel formation, produce fibrochondrocytes, inducecellular infiltration into the product, induce cellular proliferation,and produce cellular and spatial organization to form athree-dimensional meniscus tissue.

The ability of the product of the present invention to repair and/orregenerate both vascular and avascular cartilage in vivo has not beenachieved by any of the presently known cartilage repairdevices/compositions or methods. Moreover, previous devices and methodshave been primarily directed to the repair of very small defects andhave not been successful in solving problems associated with repair oflarge, clinically relevant defects. Without being bound by theory, thepresent inventors believe that the reason that these previous approachesfailed to adequately repair cartilage is that they were not able torecapitulate natural cartilage ontogeny faithfully enough, this naturalontogeny being based on a very complicated system of different factors,factor combinations and factor concentrations with temporal and localgradients. A single recombinant factor or two recombinant factors maylack the inductive complexity to mimic cartilage development to asufficient degree. Similarly, the system used to provide one or tworecombinant factors may not have been able to mimic the gradientcomplexity of the natural system to a satisfactory degree or to maintaina factor concentration for a time that is sufficient to allow a full andpermanent differentiation of precursor cells to chondrocytes. Withoutbeing bound by theory, the present inventors believe that the repair ofcertain defects, particularly large defects, requires the maintenance ofa sufficient concentration of a particular complex mixture of repairfactors at the site for a time sufficient to induce the proper formationof cartilage.

One aspect of the present invention is directed to a product for repairof cartilage lesions. In one embodiment, such a product includes: (a) acartilage repair matrix; and, (b) a cartilage-inducing compositionassociated with the matrix for provision of chondrogenesis-enhancingproteins (described in detail below), whereby each of thechondrogenesis-enhancing proteins is provided as a protein and/or by arecombinant nucleic acid molecule encoding the protein operativelylinked to a transcription control sequence.

According to the present invention, the phrase “cartilage-inducingcomposition” refers to a formulation which contains a mixture ofdifferent chondrogenesis-enhancing proteins which includes: (1) at leasta first and second transforming growth factor β (TGFβ) superfamilyprotein, wherein the first TGFβ superfamily protein is different fromthe second TGFβ superfamily protein; and, (2) at least one protein froma family of proteins selected from the group of: growth factor proteinsand bone matrix proteins. In a preferred embodiment, thecartilage-inducing composition useful in the product of the presentinvention has an identifying characteristic which includes: an abilityto induce cellular infiltration, an ability to induce cellularproliferation, an ability to induce angiogenesis, and/or an ability toinduce cellular differentiation to type II collagen-producingchondrocytes, in vivo or under appropriate in vitro conditions.

According to the present invention, a mixture ofchondrogenesis-enhancing proteins having the above-defined minimalprotein components is characterized as being capable, when culturedtogether with ATDC5 cells for seven days at a concentration of about 100ng/ml or less, of inducing a statistically significant increase in A₅₉₅in an Alcian Blue assay performed with the ATDC5 cells. The specificconditions associated with such an ATDC5/Alcian Blue assay are describedin detail below. It is noted that although the mixture ofchondrogenesis-enhancing proteins has the above-describedcharacteristic, an individual chondrogenesis-enhancing protein, whenisolated from the other proteins in the mixture, is not necessarilychondrogenic. For example, as described below, a bone matrix proteinsuch as osteocalcin is a chondrogenesis-enhancing protein according tothe present invention, because when such protein is combined with atleast two TGFβ superfamily proteins, the mixture of proteins is capableof inducing a significant increase in A₅₉₅ in an ATDC5 Alcian Blueassay. Osteocalcin is not, however, chondrogenic in the absence of thetwo TGFβ superfamily proteins.

According to the present invention, the chondrogenesis-enhancingproteins in the cartilage-inducing composition of the present inventioninclude at least two different members of the TGFβ superfamily proteins.In a preferred embodiment, the chondrogenesis-enhancing proteins includeat least three different members of the TGFβ superfamily proteins, andin increasing preference, at least four, five, six, seven, eight, nine,and most preferably ten different members of the TGFβ superfamilyproteins. As used herein, a “TGFβ superfamily protein” can be anyprotein of the art-recognized superfamily of extracellular signaltransduction proteins that are structurally related to TGFβ1-5.Preferably, a TGFβ superfamily protein suitable for use in the presentinvention is selected from the following proteins: TGFβ1, TGFβ2, TGFβ3,bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9, cartilage-derived morphogenetic protein (CDMP)-1, CDMP-2,and/or CDMP-3. More preferably, the chondrogenesis-enhancing proteins ofthe present invention include at least two of: TGFβ1, TGFβ2, TGFβ3,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP-1, CDMP-2, and/or CDMP-3.In one embodiment, the at least first and second TGFβ superfamilyproteins in a cartilage-inducing composition of the present inventionare selected from at least one of BMP2-8, and more preferably, at leastone of TGFβ1-5, and even more preferably, at least one of CDMP1-3.

According to the present invention, the chondrogenesis-enhancingproteins in the cartilage-inducing composition of the present inventioninclude, in addition to at least two different TGFβ superfamilyproteins, at least one bone matrix protein or at least one growth factorprotein. In a preferred embodiment, the chondrogenesis-enhancingproteins include at least one bone matrix protein and at least onegrowth factor protein. In a more preferred embodiment, thechondrogenesis-enhancing proteins include, in increasing preference, atleast two, three, four, and most preferably five different bone matrixproteins, and/or at least two growth factor proteins.

As used herein, “bone matrix proteins” are any of a group of proteinsknown in the art to be a component of or associated with the minutecollagenous fibers and ground substances which form bone matrix. As usedherein, a bone matrix protein is not a member of the TGFβ superfamily asdescribed herein, nor a growth factor protein as described herein. Bonematrix proteins can include, but are not limited to, osteocalcin,osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L pre,osteopontin, matrix GLA protein (MGP), biglycan, decorin,proteoglycan-chondroitin sulfate III (PG-CS III), bone acidicglycoprotein (BAG-75), thrombospondin (TSP) and/or fibronectin.Preferably, bone matrix proteins suitable for use with the product ofthe present invention include one or more of: osteocalcin, osteonectin,MGP, TSP, BSP, lysyloxidase and cathepsin L pre. In one embodiment, theat least one bone matrix protein includes osteocalcin, osteonectin, BSP,lysyloxidase and cathepsin L pre. A particularly preferred bone matrixprotein is MGP, and more preferred is osteonectin, and most preferred isTSP.

As used herein, “growth factor proteins” are any of a group of proteinscharacterized as an extracellular polypeptide signaling molecule thatstimulates a cell to grow or proliferate. Such growth factors may alsohave other actions besides the induction of cell growth orproliferation. As used herein, a growth factor is not a member of theTGFβ superfamily as defined herein nor is it a bone matrix protein asdefined herein. Preferably, growth factor proteins suitable for use withthe product of the present invention include one or more of: fibroblastgrowth factor I (FGF-I), FGF-II, FGF-9, leukocyte inhibitory factor(LIF), insulin, insulin growth factor I (IGF-I), IGF-II,platelet-derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromalderived factor-2 (SDF-2), pituitary thyroid hormone (PTH), growthhormone, hepatocyte growth factor (HGF), epithelial growth factor (EGF),transforming growth factor-α (TGFα) and hedgehog proteins. A mostpreferred growth factor protein for use with the product of the presentinvention is FGF-I.

In one embodiment of the present invention, the mixture ofchondrogenesis-enhancing proteins can also include one or more serumproteins. As used herein, serum proteins are any of a group of proteinsthat is known to be a component of serum. A serum protein is not amember of the TGFβ superfamily, a bone matrix protein or a growthfactor, as described herein. Preferably, the chondrogenesis-enhancingproteins include, in increasing preference, at least one, two, three,and most preferably four different serum proteins. Serum proteinssuitable for use with the product of the present invention include oneor more of albumin, transferrin, α2-Hs GlycoP, IgG, α1-antitrypsin,β2-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb. Preferably,serum proteins suitable for use with the product of the presentinvention include one or more of albumin, transferrin, Apo A1 LP andFactor XIIIb.

In one embodiment of the present invention, a mixture ofchondrogenesis-enchancing proteins suitable for use in a cartilagerepair product of the present invention includes the following proteins:TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP,FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, and cathepsin Lpre. In another embodiment, a suitable mixture ofchondrogenesis-enchancing proteins includes the following proteins:TGFβ1, TGFβ2, TGFβ3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP,FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, cathespin Lpre, albumin, transferrin, Apo A1 LP and Factor XIIIb. In yet anotherembodiment, a suitable mixture of chondrogenesis-enchancing proteinsincludes the mixture of proteins referred to herein as bone protein(BP), which is defined herein as partially-purified protein mixture frombovine long bones as described in Poser and Benedict, WO 95/13767,incorporated herein by reference in its entirety. As described in Poserand Benedict, WO 95/13767: “Bone growth factor was isolated rom thecortical diaphyses of bovine long bones. The marrow and soft tissue wascleaned from the long bones, and the bones were pulverized anddemineralized in 1.0 normal (N) hydrochloric acid at a 1:13 weight tovolume ratio for 16 hours at 25° C. The bone particles were washed indistilled water and then extracted in a buffered solution comprising of4 N guanidine hydrochloride buffered with 0.1 N Tris, pH 7.6 at aconcentration of 3 milliliters of buffered solution per gram of originalpowdered bone. The bone was extracted for 48 h at 15° C. The extractedbone particles were then passed through a series of chromatographicpurification steps as described in U.S. application Ser. No. 07/689,459to extract bone growth factor having bone inductive effect at doses lessthan 35 microgram (μg).”

According to the present invention, the relative proportions of theproteins in the mixture of chondrogenesis-inducing proteins are anyproportions which are sufficient for the mixture, at a concentration of100 ng/ml or less, to induce a statistically significant increase inA₅₉₅ in an Alcian Blue assay performed with ATDC5 cells as describedbelow. In one embodiment, the percentage of TGFβ superfamily memberswithin the mixture ranges between about 0.1% to about 50% of the totalmixture, and more preferably, between about 0.5% and about 25%, and evenmore preferably, between about 1% and about 10% of the total mixture.The percentage of growth factors within the mixture ranges between about0.01% to about 50% of the total mixture, and preferably, between about0.05% and about 25%, and even more preferably, between about 0.1% andabout 10% of the total mixture. The percentage of serum and bone matrixprotein components, either separately or combined, ranges between about20% to about 98%, and preferably between about 40% to about 98%, andeven more preferably between about 80% to about 98%. In anotherembodiment of the invention, the mixture of chondrogenesis-inducingproteins contains at least BMP-3, BMP-2 and TGFβ1, wherein the quantityof BMP-3 in the mixture is about 2-6 fold greater than the quantity ofBMP-2 and about 10-30 fold greater than the quantity of TGFβ1 in themixture.

In one embodiment of the cartilage repair product of the presentinvention, each of the chondrogenesis-enhancing proteins in thecartilage-inducing composition is provided by the composition either:(1) directly as a protein that is associated with the matrix, or (2) asa recombinant nucleic acid molecule associated with the matrix, suchrecombinant nucleic acid molecule encoding the protein and beingoperatively linked to a transcription control sequence such that theprotein can be expressed under suitable conditions. Therefore, acartilage-inducing composition of the present invention can includeproteins, recombinant nucleic acid molecules, or a combination ofproteins and recombinant nucleic acid molecules, such compositionproviding the chondrogenesis-enhancing proteins described above.

According to the present invention, a chondrogenesis-enhancing proteincan be obtained from its natural source, produced using recombinant DNAtechnology, or synthesized chemically. As used herein, achondrogenesis-enhancing protein can be a full-length protein (i.e., inits full-length, naturally occurring form), any homologue of such aprotein, any fusion protein containing such a protein, or any mimetopeof such a protein. The amino acid sequences for chondrogenesis-enhancingproteins disclosed herein, as well as nucleic acid sequences encodingthe same are known in the art and are publicly available, for example,from sequence databases such as GenBank. Such sequences can therefore beobtained and used to produce proteins and recombinant nucleic acidmolecules of the present invention.

A homologue is defined as a protein in which amino acids have beendeleted (e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). A homologue of achondrogenesis-enhancing protein is a protein having an amino acidsequence that is sufficiently similar to a naturally occurringchondrogenesis-enhancing protein amino acid sequence that the homologuehas substantially the same or enhanced biological activity compared tothe corresponding naturally occurring protein.

As used herein, a mimetope (also referred to as a synthetic mimic) of achondrogenesis-enhancing protein according to the present inventionrefers to any compound that is able to mimic the activity of such achondrogenesis-enhancing protein, often because the mimetope has astructure that mimics the chondrogenesis-enhancing protein. Mimetopescan be, but are not limited to: peptides that have been modified todecrease their susceptibility to degradation; anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceousimmunogenic portions of an isolated protein (e.g., carbohydratestructures); and synthetic or natural organic molecules, includingnucleic acids. Such mimetopes can be designed using computer-generatedstructures of naturally occurring chondrogenesis-enhancing protein.Mimetopes can also be obtained by generating random samples ofmolecules, such as oligonucleotides, peptides or other organic orinorganic molecules, and screening such samples by affinitychromatography techniques using the corresponding binding partner.

According to the present invention, a fusion protein is a protein thatincludes a chondrogenesis-enhancing protein-containing domain attachedto one or more fusion segments. Suitable fusion segments for use withthe present invention include, but are not limited to, segments thatcan: enhance a protein's stability; enhance the biological activity of achondrogenesis-enhancing protein; and/or assist purification of achondrogenesis-enhancing protein (e.g., by affinity chromatography). Asuitable fusion segment can be a domain of any size that has the desiredfunction (e.g., imparts increased stability, imparts enhanced biologicalactivity to a protein, and/or simplifies purification of a protein).Fusion segments can be joined to amino and/or carboxyl termini of thechondrogenesis-enhancing protein-containing domain of the protein andcan be susceptible to cleavage in order to enable straight-forwardrecovery of a chondrogenesis-enhancing protein. Fusion proteins arepreferably produced by culturing a recombinant cell transformed with afusion nucleic acid molecule that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of achondrogenesis-enhancing protein-containing domain. Preferred fusionsegments include a metal binding domain (e.g., a poly-histidinesegment); an immunoglobulin binding domain (e.g., Protein a; Protein G;T cell; B cell; Fc receptor or complement protein antibody-bindingdomains); a sugar binding domain (e.g., a maltose binding domain);and/or a “tag” domain (e.g., at least a portion of β-galactosidase, astrep tag peptide, other domains that can be purified using compoundsthat bind to the domain, such as monoclonal antibodies).

Preferably, a mixture of chondrogenesis-enhancing proteins according tothe present invention is capable, when cultured together with ATDC5cells for seven days at a concentration of about 100 ng/ml or less, ofinducing a statistically significant increase in A₅₉₅ in an Alcian Blueassay performed with the ATDC5 cells. In a preferred embodiment, amixture of chondrogenesis-enhancing proteins is capable of inducing asignificant increase in A₅₉₅ in an Alcian Blue assay performed with theATDC5 cells when cultured under the above conditions at a concentrationof about 50 ng/ml or less, and more preferably, about 25 ng/ml or less,and even more preferably, about 10 ng/ml or less. As used herein, astatistically significant increase is defined as an increase in A₅₉₅ ascompared to a control, in which the probability of such an increasebeing due to chance is p<0.05, and more preferably, p<0.001, and evenmore preferably, p<0.005.

According to the present invention, an ATDC5 Alcian Blue assay is knownin the art and is described, for example, in von Schroeder et al., 1994,Teratology 50:54-62. For the purposes of determining whether a mixtureof chondrogenesis-enhancing proteins meets the requirements of beingcapable of inducing a significant increase in A₅₉₅ in an Alcian Blueassay performed with the ATDC5 cells when cultured with such cells at agiven concentration (e.g., 100 ng/ml), the following protocol can beused.

Murine ATDC5 cells were deposited by T. Atsumi (Deposit No. RCB0565) andare publicly available from the Riken Cell Bank, 3-1-1 Koyadai, TsukubaScience City, 305 Japan. The ATDCS cells are maintained in 100×20 mmstandard tissue culture plates in Dulbecco's modified Eagle's medium(DMEM):Ham's F-12 (1:1) media that contains 5% fetal bovine serum,penicillin (50 U/ml), and streptomycin (50 mg/ml). Cultures areincubated in a humidified incubator at 37° C. and 5% CO₂. Passages 3-8can be used to assay the activity of the mixture of proteins to beevaluated. To perform the Alcian Blue assay, first, the micromassculture technique is performed as described in Atkinson et al., 1997,ibid., with minor alterations. Briefly, trypsinized cells areresuspended in the ATDC5 culture medium described above at aconcentration of about 100,000 cells/25 μl. The 25 μl spot of cells isplaced in the center of a 24 well polystyrene microtiter tissue culturedish. After 1.5 hours, 1 ml of the culture media described above isadded to the dish. After overnight incubation at 37° C. and 5% CO²,media containing various concentrations of the mixture ofchondrogenesis-inducing proteins (e.g., 100 ng/ml, 50 ng/ml, 25 ng/ml,10 ng/ml), 5% FBS, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphateare added (Day 0), and the incubation is continued. This latter media isthen replaced every 3-4 days (for a total of 2 more additions of BP).After incubation with the mixture of proteins to be tested, on Day 7,the culture media is removed and the cultures are washed three timeswith 1 ml of PBS. The cultures are then fixed with 10% neutral bufferedformalin for 15 hours and washed twice with 0.5 N HCl. Cultures arestained for one hour at room temperature with a 0.5% Alcian Bluesolution (pH 1.4). The stain is then removed and the cultures are washedwith PBS to remove unbound stain. The blue stain is then extracted withguanidium HCl (4M, pH 1.7) at 70° C. for 18 hours, followed bymeasurement of absorption at 595 nm.

It is noted that those of skill in the art will be able to make minormodifications to the above protocol and obtain a similar outcome. Suchmodifications include seeding the bottom of a well with ATDC5 cells(i.e, about 25,000-50,000, not in micromass culture), using a serumsubstitute in the media, altering the concentration of serum, and/oromitting ascorbic acid and/or β-glycerophosphate from the media. Minormodifications to the Alcian Blue assay itself can include altering thepH of the Alcian Blue solution within the range from about pH 1 to aboutpH 1.4 and altering the concentration of the Alcian Blue solution withinthe range from about 0.05% to about 0.5%.

As discussed above, one or more of the chondrogenesis-enhancing proteinsin the cartilage-inducing composition can be provided by the compositionas a recombinant nucleic acid molecule associated with the cartilagerepair matrix, such recombinant nucleic acid molecule encoding achondrogenesis-enhancing protein and being operatively linked to atranscription control sequence such that the protein can be expressedunder suitable conditions. A recombinant nucleic acid molecule useful inthe present invention can include an isolated natural gene encoding achondrogenesis-enhancing protein or a homologue of such a gene, thelatter of which is described in more detail below. A nucleic acidmolecule useful in the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated nucleic acid molecule encoding achondrogenesis-enhancing protein can be isolated from its natural sourceor produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules can include, for example, natural allelicvariants and nucleic acid molecule homologues modified by nucleotideinsertions, deletions, substitutions, and/or inversions in a manner suchthat the modifications do not substantially interfere with the nucleicacid molecule's ability to encode a chondrogenesis-enhancing protein ofthe present invention or to form stable hybrids under stringentconditions with natural gene isolates. An isolated nucleic acid moleculecan include degeneracies. As used herein, nucleotide degeneracies refersto the phenomenon that one amino acid can be encoded by differentnucleotide codons. Thus, the nucleic acid sequence of a nucleic acidmolecule that encodes a chondrogenesis-enhancing protein of the presentinvention can vary due to degeneracies.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., ibid.). For example, nucleic acid molecules can be modified using avariety of techniques including, but not limited to, by classicmutagenesis and recombinant DNA techniques (e.g., site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments and/or PCR amplification), or synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected by hybridization with a naturallyoccurring gene or by screening for the function of a protein encoded bythe naturally occurring nucleic acid molecule. Although the phrase“nucleic acid molecule” primarily refers to the physical nucleic acidmolecule and the phrase “nucleic acid sequence” primarily refers to thesequence of nucleotides on the nucleic acid molecule, the two phrasescan be used interchangeably, especially with respect to a nucleic acidmolecule, or a nucleic acid sequence, being capable of encoding achondrogenesis-enhancing protein.

Knowing the nucleic acid sequence encoding a naturally occurringchondrogenesis-enhancing protein according to the present inventionallows one skilled in the art to, for example, (a) make copies of thosenucleic acid molecules, and (b) obtain nucleic acid molecules includingat least a portion of such nucleic acid molecules (e.g., nucleic acidmolecules including full-length genes, full-length coding regions,regulatory control sequences, truncated coding regions). Such nucleicacid molecules can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies; traditional cloningtechniques using oligonucleotide probes to screen appropriate librariesor DNA; and PCR amplification of appropriate libraries or DNA usingoligonucleotide primers. Techniques to clone and amplify genes aredisclosed, for example, in Sambrook et al., ibid.

According to the present invention, a nucleic acid molecule encoding achondrogenesis-enhancing protein is operatively linked to one or moretranscription control sequences to form a recombinant molecule. Thephrase “operatively linked” refers to linking a nucleic acid molecule toa transcription control sequence in a manner such that the molecule isable to be expressed when transfected (i.e., transformed, transduced ortransfected) into a host cell.

Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells useful in the product and method of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in mammalian, bacterial, insect cells, andpreferably in mammalian cells.

One or more recombinant nucleic acid molecules encoding achondrogenesis-enhancing protein can be used to produce the protein. Inone embodiment, the protein is produced by expressing a recombinantnucleic acid molecule under conditions effective to produce the protein.A preferred method to produce an encoded protein is by transforming ahost cell with one or more recombinant molecules to form a recombinantcell. Suitable host cells to transform include any mammalian cell thatcan be transformed. Host cells can be either untransfected cells orcells that are already transformed with at least one nucleic acidmolecule. Host cells useful in the present invention can be any cellcapable of producing a chondrogenesis-enhancing protein. In a preferredembodiment, the host cell itself is useful in enhancing chondrogenesis.A particularly preferred host cell includes a fibrochondrocyte, achondrocyte, and a mesenchymal precursor cell.

According to the method of the present invention, a host cell can betransformed with a recombinant nucleic acid molecule encoding achondrogenesis-enhancing protein in vitro or in vivo. Transformation ofa recombinant nucleic acid molecule into a cell in vitro can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. The resulting recombinant cell canthen be associated with the cartilage repair matrix of the presentinvention by any suitable method to provide the chondrogenesis-enhancingproteins.

Recombinant nucleic acid molecules can be delivered in vivo andassociated with the cartilage repair matrix in a variety of methodsincluding, but not limited to, (a) administering a naked (i.e., notpackaged in a viral coat or cellular membrane) nucleic acid molecule(e.g., as naked DNA or RNA molecules, such as is taught, for example inWolff et al., 1990, Science 247, 1465-1468); (b) administering a nucleicacid molecule packaged as a recombinant virus or a recombinant cell(i.e., the nucleic acid molecule is delivered by a viral or cellularvehicle), whereby the virus or cell is associated with the cartilagerepair matrix; or (c) administering a recombinant nucleic acid moleculeassociated with the cartilage repair matrix via a delivery vehicle suchas a liposome or nanosphere delivery system described herein.

As discussed above, a recombinant nucleic acid molecule encoding achondrogenesis-enhancing protein can be associated with the cartilagerepair matrix as a recombinant virus particle. A recombinant virusincludes a recombinant molecule that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient. A number of recombinantvirus particles can be used, including, but not limited to, those basedon alphaviruses, poxviruses, adenoviruses, herpesviruses, andretroviruses. When administered to an animal, a recombinant virusinfects cells at the site of administration of the cartilage repairproduct and directs the production of a chondrogenesis-enhancingprotein.

Suitable liposomes for use as a delivery vehicle for a recombinantnucleic acid in vivo include any liposome. Preferred liposomes of thepresent invention include those liposomes commonly used in, for example,gene delivery methods known to those of skill in the art. Methods forpreparation of liposomes and complexing nucleic acids with liposomes arewell known in the art.

As described in detail below, a cartilage-inducing composition isassociated with a cartilage repair matrix, such that the cartilagerepair matrix serves, in one capacity, as a delivery vehicle for thecomposition to be delivered to the site of a cartilage lesion. Suitablemethods for associating a cartilage-inducing composition containingchondrogenesis-enhancing proteins and/or recombinant nucleic acidmolecules encoding such proteins with a cartilage repair matrix includeany method which allows the proteins and/or recombinant nucleic acidmolecules to be delivered to a site of cartilage repair together with acartilage repair matrix such that the cartilage repair product iseffective to repair and/or regenerate cartilage at the site. Suchmethods of association include, but are not limited to, suspension ofthe composition within the cartilage repair matrix, freeze-drying of thecomposition onto a surface of the matrix and suspension within thematrix of a carrier/delivery formulation containing the composition.Additionally, the cartilage-inducing composition can be associated withthe matrix prior to placement of the product into a cartilage lesion(i.e., the association of the composition with matrix occurs ex vivo) oralternatively, a cartilage repair matrix can first be implanted into alesion, followed by association of the cartilage-inducing compositionwith the matrix, such as by injection into or on top of the matrix(i.e., the association of the composition with matrix occurs in vivo). Acartilage-inducing composition can contain additional deliveryformulations or carriers which enhance the association of thecomposition with the matrix, which enhance the delivery of thecomposition to the appropriate cells and tissue at the site of thelesion, and which assist in controlling the release of the factors inthe composition at the site of the lesion. Suitable deliveryformulations include carriers, which, as used herein, include compoundsthat increase the half-life of a cartilage-inducing composition in thetreated animal. Suitable carriers include, but are not limited to,polymeric controlled release vehicles, biodegradable implants,liposomes, bacteria, viruses, oils, cells, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a cartilage-inducing composition of the presentinvention in a controlled release vehicle. Suitable controlled releasevehicles include, but are not limited to, biocompatible polymers, otherpolymeric matrices, capsules, microcapsules, microparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheres,and transdermal delivery systems. Other controlled release formulationsof the present invention include liquids that, upon association with thematrix or upon administration to an animal, form a solid or a gel insitu. Such controlled release vehicles are preferably associated withthe cartilage repair matrix by one of the above-described methods.Preferred controlled release formulations are biodegradable (i.e.,bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention at the siteof a cartilage lesion of a treated animal at a constant rate sufficientto attain therapeutic dose levels of the chondrogenesis-enhancingproteins provided by the composition to result in enhancement ofchondrogenesis at the lesion. A particularly preferred controlledrelease vehicle according to the present invention is a nanospheredelivery vehicle.

A nanosphere delivery vehicle according to the present inventionincludes the nanosphere delivery vehicle described in copending PCTApplication No. PCT/EP 98/05100, which is incorporated herein byreference in its entirety. In a preferred embodiment, such a deliveryvehicle includes polymer particles having a size of less than 1000 nmand being loaded with between 0.001% and 17% by weight of thecartilage-inducing composition. The nanospheres have an in vitroanalytically determined release rate profile with an initial burst ofabout 10% to about 20% of the total amount of the composition over afirst 24 hour period, and a long time release rate of a least 0.1% perday during at least seven following days.

A cartilage-inducing composition useful in the cartilage repair productof the present invention can also include one or more pharmaceuticallyacceptable excipients. As used herein, a pharmaceutically acceptableexcipient refers to any substance suitable for associating acartilage-inducing composition with a cartilage repair matrix andmaintaining and delivering the components of the composition (e.g.,proteins and/or recombinant nucleic acid molecules) to the appropriatecells at a suitable in vivo site (i.e., a cartilage lesion). Preferredpharmaceutically acceptable excipients are capable of maintaining anucleic acid molecule in a form that, upon arrival of the nucleic acidmolecule at the delivery site, the nucleic acid molecule is capable ofexpressing a chondrogenesis-enhancing protein either by being expressedby a recombinant cell or by entering a host cell at the site of thelesion and being expressed by the cell. A suitable pharmaceuticallyacceptable excipient is capable of maintaining a protein in a form that,upon arrival of the protein at the delivery site, the protein isbiologically active such that chondrogenesis at the site is enhanced.Examples of pharmaceutically acceptable excipients include, but are notlimited to water, phosphate buffered saline, Ringer's solution, dextrosesolution, serum-containing solutions, Hank's solution, other aqueousphysiologically balanced solutions, oils, esters and glycols. Aqueouscarriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity. Particularly preferredexcipients include non-ionic diluents, with a preferred non-ionic bufferbeing 5% dextrose in water (DW5). Suitable auxiliary substances include,for example, sodium acetate, sodium chloride, sodium lactate, potassiumchloride, calcium chloride, and other substances used to producephosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliarysubstances can also include preservatives, such as thimerosal, m- oro-cresol, formalin and benzol alcohol. Cartilage-inducing compositionsof the present invention can be sterilized by conventional methodsand/or lyophilized.

A cartilage-inducing composition is present in the cartilage repairproduct of the present invention at a concentration that is effective toinduce, at the site of a cartilage lesion, one or more of: cellularinfiltration, cellular proliferation, angiogenesis, and cellulardifferentiation to type II collagen-producing chondrocytes. Preferably,a cartilage-inducing composition is present in the cartilage repairproduct of the present invention at a concentration that is effective toinduce cartilage repair and/or regeneration at the site of a cartilagelesion. When the chondrogenesis-enhancing proteins are provided by thecartilage-inducing protein directly as a protein, the cartilage-inducingcomposition is typically provided at a concentration of from about 0.5%to about 33% by weight of the cartilage repair product. More preferably,the cartilage-inducing composition is provided at a concentration offrom about 1% to about 20% by weight of the cartilage repair product.When one or more of the chondrogenesis-enhancing proteins are providedby the composition as a recombinant nucleic acid molecule, anappropriate concentration of a nucleic acid molecule expressing onechondrogenesis-enhancing protein is an amount which results in at leastabout 1 pg of protein expressed per mg of total tissue protein at thesite of delivery per μg of nucleic acid delivered, and more preferably,an amount which results in at least about 10 pg of protein expressed permg of total tissue protein per μg of nucleic acid delivered; and evenmore preferably, at least about 50 pg of protein expressed per mg oftotal tissue protein per μg of nucleic acid delivered; and mostpreferably, at least about 100 pg of protein expressed per mg of totaltissue protein per μg of nucleic acid delivered. One of skill in the artwill be able to adjust the concentration of proteins and/or nucleic acidmolecules in the composition depending on the types and number ofproteins to be provided by the composition, and the delivery vehicleused.

In another embodiment of the cartilage repair product of the presentinvention, a cartilage-inducing composition can also contain a factorthat non-covalently attaches to one or more of any of thechondrogenesis-enhancing proteins or recombinant nucleic acid moleculesin the composition and thus, modify the release rate of the factor. Suchfactors include, but are not limited to, any ground substance or otherpolymeric substance. As used herein, a ground substance is defined asthe non-living matrix of connective tissue, which includes naturalpolymers and proteoglycans. Natural polymers include, but are notlimited to collagen, elastin, reticulin and analogs thereof.Proteoglycans include, but are not limited to anyglycosaminoglycan-containing molecules, and include chondroitin sulfate,dermatan sulphate, heparan sulphate, keratan sulphate and hyaluronan.Preferred ground substances include, but are not limited to, type Icollagen, type II collagen, type III collagen, type IV collagen andhyaluronic acid. Preferred other polymeric substances include, but arenot limited to, poly(lactic acid) and poly(glycolic acid).

In a further embodiment, the cartilage-inducing composition can includeone or more types of cells which are provided to further enhancechondrogenesis at the site of the cartilage lesion. Such cells include,but are not limited to, fibrochondrocytes, chondrocytes, mesenchymalprecursors, and any other cell that can serve as a chondrocyteprecursor. Such cells can be associated with the composition and thematrix by any of the methods described above. In one embodiment, atleast some of the cells are transformed with a recombinant nucleic acidmolecule encoding a chondrogenesis-enhancing protein to form arecombinant cell.

The cartilage repair product of the present invention also includes acartilage repair matrix. The cartilage repair matrix is the component ofthe cartilage repair device which provides a vehicle for delivery of thecartilage-inducing composition to the site of a cartilage lesion and asuitable scaffold upon which cartilage repair and regeneration canoccur. In a preferred embodiment, the cartilage repair matrix isbioresorbable.

According to the present invention, a cartilage repair matrix can beformed of any material that is suitable for in vivo use, and whichprovides the above-described characteristics of a cartilage repairmatrix for use with a cartilage-inducing composition of the presentinvention. The matrix can be formed of materials which include, but arenot limited to, synthetic polymers and/or a ground substance. Preferredground substances include natural polymers and proteoglycans. Naturalpolymers include, but are not limited to collagen, elastin, reticulinand analogs thereof. Proteoglycans include, but are not limited to, anyglycosaminoglycan-containing molecules. Particularly preferredglycosaminoglycans include chondroitin sulfate, dermatan sulphate,heparan sulphate, keratan sulphate and hyaluronan. Other preferredground substances include, but are not limited to, type I collagen, typeII collagen, type III collagen, type IV collagen and hyaluronic acid.Preferred synthetic polymers include poly(lactic acid) and poly(glycolicacid).

In one embodiment of the present invention, the cartilage repair matrixincludes collagen. Preferably, the matrix contains from about 20% toabout 100% collagen by dry weight of the matrix, and more preferably,from about 50% to about 100% collagen by dry weight of the matrix, andeven more preferably, from about 75% to about 100% collagen by dryweight of the matrix. In one embodiment, a suitable cartilage repairmatrix includes collagen from bovine tendon.

A cartilage repair matrix suitable for use in the present invention caninclude a material as described above which is in any suitable form foruse in repairing a cartilage lesion, including a sponge, a membrane, afilm or a gel. In one embodiment, a suitable cartilage repair matrixincludes autograft tissue, allograft tissue and/or xenograft tissue.

A cartilage repair product of the present invention is useful forrepairing a variety of defects in cartilage, including both tears andsegmental defects in both vascular and avascular cartilage tissue. Theproduct is particularly useful for repairing defects in hyaline (e.g.,articular) and/or fibrocartilage (e.g., meniscal). Examples of varioustypes of cartilage tears and segmental defects for which the cartilagerepair product of the present invention can be used are illustrated inFIGS. 1-4. Briefly, FIG. 1 shows a meniscal radial tear (FIG. 1A); ameniscal triple bucket handle tear (FIG. 1C); and a longitudinal tear inthe avascular area of a meniscus (FIG. 4A). A meniscal segmental lesionis illustrated in FIGS. 2A and 2B. FIG. 3A additionally schematicallyillustrates a cross section of the meniscus, which includes vascular,semi-vascular and avascular regions for which, prior to the presentinvention, only tears in the vascular region were repairable.

Therefore, since cartilage defects (i.e., lesions) can occur in avariety of shapes, sizes, and locations, a cartilage repair matrixsuitable for use in a cartilage repair product of the present inventionis of a shape and size sufficient to conform to a specific defect in thecartilage of the patient to be treated. Preferably, the cartilage repairmatrix, when used in the repair of a cartilage defect, achieves ageometry at the defect site that is suitable to provide a therapeuticbenefit to the patient. Such a therapeutic benefit can be anyimprovement in a patient's health and well being that is related to acorrection of the cartilage defect, and preferably, the therapeuticbenefit includes the repair of the defect such that the naturalconfiguration of the cartilage is at least partially restored.

In the case of a cartilage tear, the cartilage repair matrix istypically configured as a sheet. The sheet is preferably of a shape andsize suitable for insertion into the tear and to cover the entire tearsurface. One embodiment of a sheet type matrix is schematicallyillustrated in FIG. 3B. The use of such a matrix to repair an avascularlongitudinal tear is schematically illustrated in FIG. 4B. Preferably,the matrix provides an immediate mechanical repair of the tear, asurface for interacting the cartilage-inducing composition with thenatural cartilage tissue, and a scaffold upon which chondrogenesis canoccur. Additionally, the matrix preferably imparts upon the product amechanical stability sufficient to allow the product to be anchored intothe lesion. Prior to the present invention, a tear in meniscal vascularcartilage as shown in FIGS. 1A and 1C was repaired by suture repair andresection as illustrated in FIGS. 1B and 1D, respectively. Additionally,prior to the present invention, if the tear occurred in avascularcartilage tissue (or in semi-vascular tissue as shown in FIG. 3A), thetear would have been considered “irreparable”. The cartilage repairproduct of the present invention advantageously allows for the repair oftears in both avascular and vascular cartilage and when used in vascularcartilage, the product enhances the rate and quality of the repair ascompared to previously used products and methods, such as shown in FIGS.1B and 1D.

In the embodiment in which the cartilage repair matrix is configured asa sheet, the matrix preferably has a thickness of from about 0.1 mm toabout 3 mm, and more preferably, from about 0.5 mm to about 2 mm. Thethickness can, of course, be varied depending on the configuration ofthe tear which is to be repaired. In this embodiment, the matrix can beprepared by applying an aqueous dispersion of matrix material into amold, for example, wherein the mold sets the appropriate thickness forthe sheet. Such a method is described in Example 4. In a preferredembodiment, the matrix is prepared from an aqueous dispersion of fromabout 0.2% to about 4% collagen by weight, and more preferably, thematrix is prepared from an aqueous dispersion of from about 0.5% toabout 3% collagen by weight.

In the case of a segmental defect in cartilage (i.e., any defect that islarger and of a different shape than a tear), the cartilage repairmatrix is typically configured to achieve a suitable geometry thatrepairs the defect, and includes matrices which are configured toreplace damaged cartilage which has been removed. Prior to the presentinvention, repair of a segmental defect or indeed, any defect whichoccurred in the avascular region of meniscal tissue, typically involvedthe removal of the damaged tissue, such as by partial or completeexcision of the meniscus (i.e., a meniscectomy). Excision was thensometimes followed by a replacement prosthetic meniscus, but until thepresent invention, such methods were unable to regenerateendogenous-type cartilage in the avascular region of the meniscus. Thecartilage repair matrix useful for segmental cartilage defects (i.e.,“non-tear” defects) is preferably of a shape and size suitable forproviding an immediate mechanical repair of the defect, a surface forinteracting the cartilage-inducing composition with the naturalcartilage tissue, and a scaffold upon which chondrogenesis can occur.Additionally, the matrix preferably imparts upon the product amechanical stability sufficient to allow the product to be anchored intothe lesion. A cartilage repair matrix suitable for use for repairingsegmental defects in the cartilage repair product of the presentinvention is described in detail in U.S. Pat. No. 5,681,353 to Li et al.which is incorporated herein by reference in its entirety. Otherpreferred cartilage repair matrices are described in the Examplessection. In one embodiment, a cartilage repair matrix used to repair asegmental defect in meniscal cartilage contains a porous groundsubstance composite which includes collagen and has the shape andmechanical characteristics suitable to repair a meniscus lesion.

In one embodiment, a cartilage repair matrix suitable for use in therepair of segmental defects has a tapered shape. Such a matrix typicallyvaries in thickness from about 0.5 mm to about 3 mm at its thinnestregion to from about 4 mm to about 10 mm at its thickest region. Such amatrix typically has a density of from about 0.07 to about 0.5 gramsmatrix per cm³, and more preferably, from about 0.1 to about 0.25 gramsmatrix per cm³, wherein g/cm³ represents the number of grams in a cubiccentimeter of matrix. FIGS. 2A and 2B illustrate a cartilage repairmatrix configured to repair a segmental defect in a meniscus.

In a preferred embodiment, the cartilage repair matrix of the presentinvention is porous, which enhances the ability of the matrix to serveas a delivery vehicle for the cartilage-inducing composition andparticularly, as a scaffold for chondrogenesis, such as by allowing forthe ingrowth of cells into the matrix. Preferably, the pore size issufficient to maintain the desired mechanical strength of the matrix,while allowing sufficient ingrowth of cells for regeneration ofcartilage at the lesion. The porosity of the matrix can vary dependingon the configuration of the matrix, but typically, the matrix has a poresize of from about 10 μm to about 500 μm. When the matrix is configuredto repair a tear defect, the pore size is typically from about 10 μm toabout 100 μm. When the matrix is configured to repair a segmentaldefect, the pore size is typically from about 50 μm to about 500 μm.

When the cartilage repair matrix is configured as a sheet, it ispreferably not cross-linked. When the cartilage repair matrix isconfigured to repair a segmental defect, however, the matrix can becross-linked, such as by artificial cross-linking methods, although suchcross-linking is not required. A cartilage repair matrix can becross-linked by any suitable agent which includes, but is not limitedto, formaldehyde, glutaraldehyde, dimethyl suberimidate, carbodiimides,multi-functional epoxides, succinimidyls, Genipin, poly(glycidyl ether),diisocyanates, acyl azide, ultraviolet irradiation, dehydrothermaltreatment, tris(hydroxymethyl)phosphine, ascorbate-copper,glucose-lysine and photo-oxidizers. In one embodiment, a cartilagerepair matrix is cross-linked with an aldehyde.

Another embodiment of the present invention relates to a product for therepair of cartilage lesions which includes: (a) a cartilage repairmatrix as described in detail above; and, (b) a cartilage inducingcomposition associated with the matrix which includes cells that havebeen cultured with a mixture of the chondrogenesis-enhancing proteins aspreviously described herein. Specifically, the mixture ofchondrogenesis-enhancing proteins include: (1) at least a first andsecond transforming growth factor β (TGFβ) superfamily protein, whereinthe first TGFβ superfamily protein is different from the second TGFβsuperfamily protein; and, (2) at least one protein from a family ofproteins selected from the group of: growth factor proteins and bonematrix proteins. The chondrogenesis-enhancing proteins have beendescribed in detail above. Preferably, the cells to be cultured with themixture of proteins are cells which are involved in chondrogenesis, andinclude, but are not limited to, fibrochondrocytes, chondrocytes,mesenchymal precursors, and any other cell that can serve as achondrocyte precursor. Such cells are preferably cultured in vitro priorto their association with a cartilage repair matrix, under conditionseffective to allow the cells to interact with the proteins and initiatechondrogenesis by the cells. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit interaction of the proteins and cells andinitiation of chondrogenesis processes by the cells. An effective,medium refers to any medium in which a cell is cultured provide such aresult. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells canbe cultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes, and petri plates. Culturing can be carried outat a temperature, pH and oxygen content appropriate for a the cell. Suchculturing conditions are within the expertise of one of ordinary skillin the art. In another aspect of this embodiment of the presentinvention, a cartilage repair matrix is cultured in vitro together withthe cells and mixture of proteins prior to implantation into a cartilagelesion in vivo. In a further embodiment, the cells that have beencultured with the mixture of proteins can be associated with thecartilage repair matrix in conjunction with additionalchondrogenesis-enhancing proteins and/or recombinant nucleic acidmolecules encoding such proteins as described above.

Another embodiment of the present invention relates to a product for therepair of vascular and avascular meniscus tears. Such a productincludes: (a) a cartilage repair matrix comprising collagen andconfigured as a sheet; and, (b) a cartilage-inducing compositionassociated with the matrix for provision of chondrogenesis-enhancingproteins. The chondrogenesis-enhancing proteins include: (i) at least afirst and second transforming growth factor β (TGFβ) superfamilyprotein, wherein the first TGFβ superfamily protein is different fromthe second TGFβ superfamily protein; and, (ii) at least one protein froma family of proteins selected from the group of growth factor proteinsand bone matrix proteins. The chondrogenesis-enhancing proteins, arecharacterized in that, when cultured together with ATDC5 cells for sevendays at a concentration of about 100 ng/ml or less, they induce astatistically significant increase in A₅₉₅ in an Alcian Blue assayperformed with the cells. Each of the chondrogenesis-enhancing proteinsis provided as a protein or by a recombinant nucleic acid moleculeencoding the protein operatively linked to a transcription controlsequence. In one embodiment, the cartilage repair matrix is formed of acollagen sponge. When the lesion to be repaired is in the avascularregion, the product can additionally include a time controlled deliveryformulation as described in detail above.

Yet another embodiment of the present invention relates to a method forrepair of cartilage lesions. The method includes the steps of implantingand fixing into a cartilage lesion a product which includes: (a) acartilage repair matrix; and (b) a cartilage-inducing compositionassociated with the matrix for provision of chondrogenesis-enhancingproteins. As described in detail above, the chondrogenesis-enhancingproteins include: (i) at least a first and second transforming growthfactor β (TGFβ) superfamily protein, wherein the first TGFβ superfamilyprotein is different from the second TGFβ superfamily protein; and, (ii)at least one protein from a family of proteins selected from: growthfactor proteins and/or bone matrix proteins. Also as described above,the chondrogenesis-enhancing proteins, when cultured together with ATDC5cells for seven days at a concentration of about 100 ng/ml or less,induce a statistically significant increase in A₅₉₅ in an Alcian Blueassay performed with the cells. Additionally, each of thechondrogenesis-enhancing proteins is provided as a protein or by arecombinant nucleic acid molecule encoding the protein operativelylinked to a transcription control sequence.

The method of the present invention is useful for repairing any of thecartilage lesions described above, including both articular and meniscalcartilage lesions, and both avascular and vascular defects. The step ofimplanting is performed using surgical techniques known in the art, andtypically involves inserting the repair product directly into the tearwhen the matrix is configured as a sheet, and involves a more complexprocess of removing damaged tissue and implanting the repair productwhen the matrix is configured to repair a segmental defect. The step offixing can include attaching the product to the cartilage at the site ofthe lesion by any means suitable for attaching a matrix as describedherein to cartilage or tissue surrounding the cartilage in vivo. Such ameans for attaching can include, but is not limited to application ofbioresorbable sutures, application of non-resorbable sutures,press-fitting, application of arrows, application of nails, orapplication of a T-fix suture anchor device. Examples 5 and 7-9 describethe method of the present invention when the cartilage repair matrix isconfigured as a sheet and used to repair meniscal and articularcartilage. Example 6 describes the method of the present invention whichis used to repair meniscal and articular cartilage when the repairmatrix is configured to repair a segmental defect. FIGS. 2A and 2Billustrate the implantation and fixation of a cartilage repair productinto a segmental defect in meniscal cartilage. FIGS. 4A and 4Bschematically illustrate the repair of a longitudinal tear in avascularmeniscal tissue using a cartilage repair product configured as a sheet.

In one embodiment of the method of the present invention, a cartilagelesion which is a segmental defect is repaired by using two cartilagerepair products of the present invention. In this embodiment, asegmental defect, and preferably a meniscal segmental defect, isrepaired by trimming damaged cartilage tissue away to form a suitableinterface for implantation of the repair devices. A first cartilagerepair product having a matrix configured as a sheet is implanted andfixed along the defect (e.g., along the meniscal rim when the defect isin vascular cartilage and this cartilage has been removed). A secondcartilage repair matrix configured to replace the segmental defect, or acartilage repair product having a matrix configured to replace thesegmental defect, is implanted and fixed to the first product configuredas a sheet. The sheet provides an interface in which cells can quicklyinfiltrate and react with the cartilage repair composition. In thisembodiment, the cartilage repair product configured as a sheet containsthe cartilage repair composition as described herein, and the cartilagerepair product configured to repair the segmental defect may or may notbe associated with the cartilage repair composition, as deemed necessaryby the surgeon. FIG. 8 schematically illustrates the concurrent use ofboth a cartilage repair product configured as a sheet and a cartilagerepair product configured to repair a segmental defect.

Another embodiment of the method of the present invention relates to amethod for repair of avascular meniscus lesions. The method includes thesteps of implanting and fixing into a cartilage lesion of the avascularregion of a meniscus a cartilage repair product as described herein,wherein the cartilage repair matrix is configured as a sheet, andwherein the cartilage repair product further includes a time controlleddelivery formulation which is associated with the matrix in conjunctionwith the cartilage-inducing composition.

Another embodiment of the method of the present invention is a methodfor enhanced repair of vascular meniscus lesions. The method includesthe steps of implanting and fixing into a cartilage lesion in thevascular region of a meniscus a cartilage repair product as describedherein, wherein the cartilage repair matrix includes collagen and isconfigured as a sheet. The present inventors have discovered that theuse of the cartilage repair product of the present invention to repairvascular lesions measurably enhances the rate of repair of the vascularlesion as compared to the rate of repair of a meniscus lesion repairedin the absence of the product. According to the present invention, ameasurable enhancement of the rate of repair is any measurableimprovement in the time between Day 0 of the repair (i.e., the day theproduct is implanted into the patient) and the day on which it isdetermined that suitable cartilage tissue growth has occurred at thelesion as compared to a vascular lesion that is repaired in the absenceof the product of the present invention. Suitable cartilage tissuegrowth is defined as an initial indication of enhanced blood vesselformation, production of fibrochondrocytes, induction of cellularinfiltration into the product, induction of cellular proliferation, andproduction of cellular and spatial organization to form athree-dimensional tissue that more nearly represents endogenouscartilage tissue from the site of the lesion. In a preferred embodiment,the product of the present invention measurably enhances the rate ofrepair of a vascular cartilage lesion, as compared to a vascular lesionthat is repaired in the absence of the product of the present invention,by at least 25%, and more preferably, by at least about 50%, and morepreferably by at least about 100%.

In addition, the use of the cartilage repair product of the presentinvention to repair vascular cartilage lesions results in a measurableenhancement of the quality of repair of the vascular lesion as comparedto the quality of repair of a lesion repaired in the absence of theproduct. A measurable enhancement in the quality of repair of thevascular lesion is defined as any measurable improvement in quality ofcartilage formation at the site of the lesion, with an improvement beingdefined as development of a more normal cartilage tissue, which can beindicated by enhanced blood vessel formation, production offibrochondrocytes, induction of cellular infiltration into the product,induction of cellular proliferation, and production of cellular andspatial organization to form a three-dimensional tissue that more nearlyrepresents endogenous cartilage tissue from the site of the lesion.

More particularly, the quality of repair of cartilage tissue accordingto the present invention can be evaluated as follows.

The quality of meniscal cartilage repair (i.e., fibrocartilagecartilage) can be evaluated by histological analysis of the tissue atthe repair site on a scale of 0 to 4 based on the following parameters:I. Histological cartilage staining in the defect, II. Cellularinfiltration into the collagen implant, and III. Integration into theendogenous meniscus. These scales according to the present invention aredefined as follows:

Score I. % of cells within implant that stain with Azure at pH = 1 475-100 3 50-75 2 25-50 1 10-25 0  0-10 II. % of cells that haveinfiltrated the collagen implant 4 75-100 3 50-75 2 25-50 1 10-25 0 0-10 III. % of the repaired site that integrates with the endogenousmeniscus 4 75-100 3 50-75 2 25-50 1 10-25 0  0-10

Preferably, a measurable enhancement in the quality of repair of ameniscal lesion is defined as a higher score in at least one of theparameters defined above as I, II and III, with a “4” the highest scorein each parameter, than a lesion repaired in the absence of the productof the present invention. More preferably, a measurable enhancement inthe quality of repair of a meniscal lesion is defined as a score of atleast 1, and more preferably at least 2 and more of at least 3, and mostpreferably at least 4 in at least one of the parameters defined above asI, II and/or III, as compared to a lesion repaired in the absence of theproduct of the present invention.

The quality of osteochondral repair (articular cartilage defects) can beevaluated by histological analysis of the tissue at the repair site on ascale of 0 to 4 based on the following parameters: I. defect fillingwith bone, II. thickness of the repaired cartilage, and III. repairedcartilage integration with the endogenous cartilage. These scalesaccording to the present invention are defined as follows:

Score I. % of defect that is filled with bone 4 75-100 3 50-75 2 25-50 110-25 0  0-10 II. % of repaired cartilage that is thickness ofendogenous cartilage 4 75-100 3 50-75 2 25-50 1 10-25 0  0-10 III. % ofthe repaired cartilage that integrates with the endogenous cartilage 475-100 3 50-75 2 25-50 1 10-25 0  0-10

Preferably, a measurable enhancement in the quality of repair of anosteochondral lesion is defined as a higher score in at least one of theparameters defined above as I, II and III, with “4” being the highestscore, than a lesion repaired in the absence of the product of thepresent invention. More preferably, a measurable enhancement in thequality of repair of an osteochondral lesion is defined as a score of atleast 1, and more preferably at least 2 and more preferably at least 3,and most preferably at least 4 in at least one of the parameters definedabove as I, II or III, as compared to a lesion repaired in the absenceof the product of the present invention.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, a protein refers to one or more proteins,or to at least one protein. As such, the terms “a” (or “an”), “one ormore” and “at least one” can be used interchangeably herein. It is alsoto be noted that the terms “comprising”, “including”, and “having” canbe used interchangeably.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example demonstrates that a naturally derived mixture ofproteins isolated from demineralized bovine bones (BP) induces spheroidformation and chondrogenesis in vitro in the mesenchymal precursor celllines, 10T1/2 and C₂C₁₂.

Murine C3H/10T1/2 (ATCC No. CCL-226) embryonic mesenchymal stem cellsand C₂C₁₂ adult myoblast cells (ATCC No. CRL-1772; derived from legmuscle) were obtained from the American Type Tissue Collection. 10T1/2and C₂C₁₂ cells were proliferated in the presence of 10% and 15% FBS,respectively. Micromass cultures were performed as follows. Briefly,trypsinized cells were resuspended in media containing FBS at aconcentration of 10⁷ cells/ml, and 10 μl of cells were placed in thecenter of a 24 well microtiter tissue culture dish. After 2-3 hours at37° C., 1 ml of DMEM (for 10T1/2) or 1:1 DMEM:F-12 (for C₂C₁₂) mediacontaining 1% Nutridoma and various concentrations of BP (prepared asdescribed in Poser and Benedict, WO 95/13767, ibid.) were added. BP waspresent for the initial 48 hours and was not subsequently added.

The results presented in Table 1 show that BP concentrations greaterthan or equal to 20 ng/ml induced spheroid formation in >90% of 10T1/2micromass cultures. In >90% of C₂C₁₂ micromass cultures, BPconcentrations greater than or equal to 100 ng/ml induce spheroidformation.

TABLE 1 BP Concentration (ng/ml) Cell 0 10 20 50 100 >100 10T1/2 −− + + + + C₂C₁₂ − − − +/− + + − = spheroid formation in 0% of micromasscultures +/− = spheroid formation in <90% of micromass cultures + =spheroid formation in >90% of micromass cultures

For C₂C₁₂ cells, the resulting spheroids were placed in Bouin's fixativefor 24 hours and histology and immunocytochemistry was performed. Themyosin F1.652 antibody was purchased from the Iowa Hybridoma Bank.

More specifically, for histology, spheroids were dehydrated and fixedfor 20 minutes in absolute methanol at 4° C. Fixed sections wereinfiltrated and polymerized using the glycol methacrylate embeddingtechnique. The polymerized plugs were then sectioned at 5 mm thicknessusing a JB-4 Sorvall microtome. Sections were mounted on silate-coatedslides and stained with 0.2% Azure II at pH 1.

For immunocytochemistry, spheroids were snap-frozen in a 100%isopentane/dry ice solution, sectioned at 5 μm thickness using aReichert-Jung cryostat, and mounted on silane-coated slides. Frozensections were then fixed in 1% paraformaldehyde for 20 min. rinsed in0.05 M Tris-Cl (pH 7.4), blocked with 1% BSA for 20 min. at roomtemperature, and incubated with either goat or mouse primary antibodyfor 1 hour at room temperature. After rinsing, the sections were blockedwith 10% normal rabbit serum for 20 min. at room temperature. Thesections were then treated with 1:2,000 biotinylated, rabbit antigoatIgG followed by incubation with a 1:100 streptavidin-conjugated alkalinephosphatase. Each incubation was for 30 min. at room temperature. Themouse-antibody treated slides were incubated with an unlabeled rabbitantimouse (rat absorbed) antibody and then incubated with an alkalinephosphatase antialkaline phosphatase antibody. Each incubation was for30 minutes at room temperature. The reaction was visualized with analkaline phosphatase substrate, New Fuchsin. The slides werecounterstained with Gill's #1 hematoxylin for 10 sec., dehydratedthrough graded alcohols, and cleared in Americlear xylene substitute.Polyclonal goat primary antibodies for type II collagen were diluted1:200 in 0.5 M Tris-HCl (pH 7.4), 1% BSA, and 1% sodium azide prior touse.

Chondrogenesis is indicated by positive staining of sulfatedproteoglycans with Azure at pH 1, morphology of a rounded cell typeencompassed by a territorial matrix, and the presence of type IIcollagen. The collagen quantity was subjectively graded in duplicatesamples with a ‘−’ representing no stain detected and increasing ‘+’sreflecting increasing amounts and intensity of stain. Samples lackingthe primary antibody received a ‘−’ score.

Table 2 shows that BP (500 ng/ml) induces chondrogenic markers in 10T1/2cells over a period of 28 days.

TABLE 2 Days in culture Marker Correlated with 2 7 14 21 28 Azure (pH 1)Cartilage/sulfated − ++ + ++ +++ proteoglycan MorphologyCartilage/rounded cells − ++ ++ ++ +++ and territorial matrix Type IICartilage − ++++ ++++ ++++ ++++ Collagen

Table 3 shows that in C₂C₁₂ cells, BP (1000 ng/ml) inhibits myosinproduction (an indicator of muscle) and induces type II collagenproduction (an indicator of cartilage) after three days in culture.

TABLE 3 Marker Indicative of: Presence of Marker Myosin Muscle − Type IIcollagen Cartilage ++++

Example 2

The following example demonstrates that a naturally derived mixture ofproteins isolated from demineralized bovine bones (BP) inhibitsmyogenesis in a dose dependent manner in C₂C₁₂ cells.

For myogenesis inhibition experiments, 25,000 C₂C₁₂ cells were seeded intriplicate to a 24 well plate in DMEM media that contained 15% FBS. Thenext day (day 0), this media was replaced with media containing 1%Nutridoma +/− various concentrations of BP. Media was replaced every 2-3days.

BP concentrations (0, 10, 20, 60, 100, 400, 1000, or 3000 ng/ml) weretested for the effect on C₂C₁₂ myotube formation. As shown in Table 4, aBP concentration of 10 ng/ml produced no morphological differences whencompared to cultures lacking BP. However, at 20 and 60 ng/ml, BPsubstantially decreased the number of myotubes. Complete myotubeinhibition was observed at BP concentrations above or equal to 100ng/ml.

TABLE 4 BP concentration (ng/ml) 0 10 20 60 100 400 1000 3000 ++++ ++++++ + − − − −

Example 3

The following example demonstrates that a naturally derived mixture ofproteins isolated from demineralized bovine bones (BP) quantitativelyinduces chondrogenesis of ATDC5 cells in a dose dependent manner.

In this experiment, 100,000 ATDC5 cells/25 μl media (DMEM:Ham's F-12(1:1); 5% FBS; 50 U/ml penicillin; 50 mg/ml streptomycin) were seeded intriplicate to a 24 well plate (micromass culture). ATDC5 cells weredeposited by T. Atusmi and are publicly available as Deposit No. RCB0565from the Riken Cell Bank, 3-1-1 Koyadai, Tsukuba Science City, 305Japan. After 1½ hour, 1 ml of the media was added. The next day, thesame media containing various concentrations of BP, 50 μg/ml ascorbicacid, 5% FBS, and 10 mM β-glycerophosphate were added (day 0). Thislatter media was replaced every 3-4 days. To those skilled in the art,slight modifications can be made to this protocol to obtain a similarresult. For example, fewer cells (e.g., 25,000-50,000) could be seededto the bottom of the well (e.g., not in micromass culture). Mediacontaining a serum substitute, such as 1% Nutridoma (BoehringerMannheim) could be used in place of serum. Also, the cultures maycontain different concentrations of serum or may contain or lack theascorbic acid and/or the β-glycerophosphate.

To measure cartilage matrix production, an Alcian Blue staining methodwas used as previously described (von Schroeder et al, 1993, ibid.) withminor modifications. Briefly, after incubation with BP as describedabove, the culture media was removed and the cultures were washed threetimes with 1 ml of PBS. The cultures were then fixed with 10% neutralbuffered formalin for 15 hours and washed twice with 0.5 N HCl. Cultureswere stained for 1 hour at room temperature with a 0.5% Alcian Bluesolution (pH 1.4). The stain was removed and the cultures were washedwith PBS to remove the unbound stain. The blue stain was then extractedwith guanidium HCl (4M, pH 1.7) at 70° C. for 18 hours, followed bymeasurement of absorption at 595 nm. The cultures were performed intriplicate. Using this method, glycosaminoglycan quantification wasdemonstrated to be proportional to ³⁵SO₄ incorporation (Lau, et al.,1993, Teratology 47:555-563).

To determine the effect of BP on chondrogenesis, 0, 10, 20, 60, 100,400, and 1000 ng/ml BP were added to chondrogenic ATDC5 micromasscultures and, after 7 days, the cultures were stained with Alcian Blue.Qualitative, microscopic evaluation showed that cultures lacking BPcontained no positive staining and cultures containing low BPconcentrations (10, 20, and 60 ng/ml) showed diffuse staining. Withhigher doses of BP (100, 400 and 1000 ng/ml BP), both staining intensityand the number of positively stained focal cell areas increased in adose dependent manner. Cultures treated with 400 and 1000 ng/ml BPcontained positively stained focal clusters consisting of rounded cellsencompassed by a territorial matrix (data not shown). Negatively stainedareas were also present. Quantitation of Alcian Blue staining (FIG. 5)revealed that BP concentrations as low as 10 ng/ml significantlyincreased Alcian Blue content compared to control cultures lacking BP(p<0.0005). Maximal Alcian Blue staining was observed at 400 ng/ml BP.

BP also stimulates chondrogenesis in cultures containing a serumsubstitute, Nutridoma, and this stimulation occurs over 14 days. In theabsence of BP, very little Alcian Blue staining was observed at days 7and 14 (FIG. 6). However, cultures containing 1000 ng/ml BPsignificantly (p<0.001) stimulated chondrogenesis 8.5 and 11.2 fold atdays 7 and 14, respectively, compared to cultures lacking BP (FIG. 6).

To determine whether other fractions isolated from bone have a similaractivity as BP, the following experiment was performed. BP was purifiedas previously described (Poser and Benedict, PCT Publication No.WO95/13767). However, after the BP containing fractions were collectedfrom the HPLC, two fractions, IBP and PIBP, that were more hydrophobicthan BP, were also collected. The chondrogenic activity of BP, IBP andPIBP was tested on ATCD5 cells. FIG. 7 shows that both IBP and PIBPinduce significantly less (p<0.005) chondrogenesis than BP.

To determine whether different lots of BP differed significantly forchondrogenesis, two lots were tested at two concentrations. Table 5shows that the chondrogenic activity is not significantly differentbetween two different BP lots.

TABLE 5 Absorbance BP Lot # 200 ng/ml 300 ng/ml 97182 0.364 +/− 0.0190.535 +/− 0.031 98001 0.353 +/− 0.041 0.484 +/− 0.043

Example 4

The following example describes a procedure for preparation of acartilage repair product of the present invention, configured as asheet.

Bovine tendon type I collagen, obtained from ReGen Biologics, Inc., wasplaced in one syringe. The appropriate volume of 10 mM acetic acid wasplaced in another syringe. For sponges that contain BP, the appropriatedose of BP was placed in the syringe containing 10 mM acetic acid. Thesyringes were coupled, and the contents of each syringe were mixed toproduce a 2% collagen (w/w) slurry. After an overnight incubation, thepreparation was placed in molds of appropriate thickness, frozen at −20°C. for more than 4 hours, and lyophilized until dry. Approximate implantdimensions for the sheet are length: 15.5 mm; width: 4.8 mm; andthickness: 1.2 mm.

Example 5

The following example describes the procedure for surgical implantationof a cartilage repair product of the present invention which isconfigured as a sheet.

Adult castrated mail Capra hircus goats weighing 50-70 pounds were usedin this study. After pre-anesthesia administration, the goats wereanesthetized with an inhalation anesthetic (IsoFlurane). The hind limbsof each goat were clipped, scrubbed, and draped in preparation for thesurgical procedure. An incision was made over the medial aspect of theknee (stifle) joint. The sartorial fascia was dissected to expose themedial collateral ligament (MCL). A wedge shaped femoral bone blockcentered on the MCL was created with an oscillating saw and osteotome.The block was then drilled and tapped for later reattachment using a 3.5mm bicortical screw. The bone block was then elevated to expose thesurface of the medial meniscus leaving the coronary ligaments intact. Alongitudinal tear was placed in the central, avascular portion of themeniscus using a scalpel. Four treatments were tested in the defect asfollows. Group I: nothing was placed in the defect, but the defect wasrepaired using 1-0 non-absorbable sutures and a horizontal mattresstechnique (i.e., a conventional repair method); Group II: a collagensheet was sutured into the defect using the method of Group I; and,Group III: a collagen sheet containing 35 μg BP was sutured into thedefect using the method of Group I.

After reduction and fixation of the bone block with a screw and washer,the sutures were tied outside the capsulate. The subcutaneous tissueswere then closed with absorbable suture and subcuticular 3-0 proleneskin closure. After recovery from anesthesia, the goats were placed inholding pens and then placed in an outdoor facility with unrestrictedactivity.

After 8 weeks, the animals were euthanized with a pentobarbitol sodiumsolution (100 mg/kg IV) solution. The menisci were fixed in a 10%neutral buffered formalin solution. Tissue sections for the menisci werethen stained with H+E.

Group III defects that contained 35 μg BP were filled with tissue fromthe superior to inferior sides of the meniscus and the reparative tissuewas integrated into the endogenous meniscus tissue. In addition, theendogenous meniscus tissue immediately adjacent to the defect was morecellular than normal endogenous meniscus, indicating a reparativeresponse. In contrast, Group I and II defects remained empty; noevidence of healing was observed.

Example 6

The following example describes the procedure for surgical implantationof a cartilage repair product of the present invention which isconfigured to replace a segmental meniscal cartilage defect.

If a meniscal repair can not be accomplished (i.e., such as by using aproduct of the present invention configured as a sheet) due to theseverity of the tear or poor quality of the tissue, then preparation ofthe meniscal rim is undertaken by removing the torn portions of thecartilaginous tissue. A cartilage repair product of the presentinvention can be configured to replace the segmental meniscal cartilagedefect, thereby serving to both regenerate and repair meniscalcartilage. The surgical procedure, in the absence of the cartilagerepair product of the present invention, has been previously describedby Stone and Rosenberg (cites). The following procedure describes amodification of the procedure of Stone and Rosenberg, incorporating theuse of a cartilage repair product of the present invention.

Briefly, the torn, fragmented pieces of native meniscal cartilage areremoved, and the attachment sites for meniscal horns are anatomicallyplaced or the natural peripheral rim and horns are preserved. Using thesurgical techniques described by Stone and Rosenberg, a cartilage repairmatrix configured as a collagen meniscus implant (CMI; having the shapeand mechanical characteristics of the meniscus), which is associatedwith a cartilage repair composition according to the present invention,is implanted and fixed to the meniscal rim. During the surgery, theperiphery of the meniscal implant must be attached securely enough topermit axial and rotational loads, and the surrounding capsule andligaments of the knee joint must be neither excessively violated norconstrained by the fixation technique.

In some cases, a cartilage repair product of the present invention whichis configured as a sheet can be fixed to the meniscus rim using the samesurgical procedures as described for the CMI product above. Thiscombination use of the cartilage repair product of the present inventionis illustrated schematically in FIG. 8. Use of such a sheet optimizesthe integration between the meniscal rim and the CMI by providing athin, porous collagen containing the cartilage repair composition. Thesheet provides an interface in which cells can quickly infiltrate andreact with the cartilage repair composition. In this embodiment, thecartilage repair product configured as a sheet contains the cartilagerepair composition of the present invention, and the cartilage repairproduct configured as a CMI may or may not be associated with thecartilage repair composition.

Example 7

The following experiment demonstrates that a cartilage repair product ofthe present invention enhances and induces meniscus regeneration in bothvascular and avascular meniscal tissue in vivo.

One hind leg was operated on in 1-2 year old female sheep. Briefly, thesurgical approach was to make an incision in the skin and subcutaneoustissues from the distal fourth of the femur distally to the proximalfourth of the tibia. A bone block that contained the origin of themedial collateral ligament was then removed from the femoral condyle.The tibia was abducted and externally rotated. Using a 3 mm dermalbiopsy punch (Miltex), two defects were created in thevascular/avascular zone of the medial meniscus. The Collagen MeniscusImplant (CMI) was obtained from ReGen Biologics. Using a 4 mm dermalbiopsy punch (Miltex), CMI sponges were cut to size. The 4 mm implantswere press-fit into the meniscal defects. CMI either lacked or containedBP (69 μg/mg collagen). To produce CMI containing BP, the followingprotocol was followed. A saturating volume of 10 mM acetic acid solutionthat contained BP was added to the 4 mm CMI sponges. The sponges wereplaced in a humidified environment at room temperature for 30 minutes,frozen at −20° C. for more than 4 hours, and then lyophilized until dry.

The animals were sacrificed after four weeks and the menisci were fixedin a 10% neutral buffered formalin solution. Tissue sections werestained with Azure B at pH 1 and 4.5. Cells infiltrated the CMI matrixboth in the presence or absence of BP, however, histology resultsdemonstrated that only undifferentiated fibroblast cells were present inthe implant that lacked BP (data not shown). In contrast, the BPimpregnated implant contained differentiated fibrochondrocytes (data notshown). Fibrochondrocytes were identified by rounded cell shape andcells surrounded by positively staining lacunae. In addition, CMIsponges that lacked BP contained cells that did not stain withantibodies against type II collagen. In contrast, CMI sponges thatcontained BP contained cells that stained positively with antibodiesagainst type II collagen.

Example 8

The following example demonstrates that a cartilage repair product ofthe present invention enhances and induces articular cartilage repair invivo.

Five 8 month old (skeletally mature) New Zealand White Rabbits were usedin this experiment. Bilateral defects, 8 mm long, 3 mm wide, and 3 mmdeep, were produced in the trochlear groove of each knee. For eachrabbit, a 2% collagen sponge 8 mm long, 3 mm wide and 3 mm deep, wasplaced in each knee defect, with one knee receiving the sponge in theabsence of BP, and one knee receiving the sponge containing 40-45 μg ofBP. Autologous fibrin was prepared from 50 ml of rabbit blood using thealcohol precipitation method according to Kjaergard et al., 1992, Surg.Gynecol. Obst. 175(1): 72-73. Approximately 20 μl of fibrinogen wasplaced on top of the collagen and was polymerized with 10 μl bovinethrombin (Gentrak). After 2 months, the animals were sacrificed.

A 500 μm slab of the defect area was embedded in glycol methacrylateafter fixation in cold methanol and stained with azure B at pH 1 and4.5. An adjacent 500 μm slab was fixed and decalcified with EDTA in 4%paraformaldehyde and embedded in paraffin. Serial sections were digestedwith chondroitinase ABC and stained with hemotoxilin and eosin.

Histologically, defects that contained the collagen sponge only producedmore immature tissue than defects that contained collagen+BP. Withoutadded BP, the bone region was filled with fibrocartilage andfibroblastic tissue. In contrast, defects containing BP were filled withosteoblasts and contained a bone morphology. The cartilage area, in theabsence of BP, was often 3 mm dep, rather than the normal 0.5-0.7 mmdeep. With BP added, the cartilage surface thickness was nearlyidentical to the endogenous cartilage thickness. In addition, polarizedlight microscopy revealed that the BP treated defects contained a moremature collagen fiber architecture than defects that did not contain BP.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art.

It is to be expressly understood, however, that such modifications andadaptations are within the scope of the present invention, as set forthin the following claims:
 1. A product for repair meniscal cartilagelesions, comprising: a. a cartilage repair matrix selected from thegroup consisting of: a matrix configured as a sheet which conforms to ameniscal radial tear, a matrix configured as a sheet which conforms to ameniscal longitudinal tear, a matrix configured as a tapered shape whichconforms to a segmental defect in a meniscus, and a matrix configured asa meniscus or a segmental portion thereof; b. a cartilage-inducingcomposition contained on or within said matrix comprising a mixture ofproteins comprising: i. transforming growth factor β (TGFβ) superfamilyproteins: TGFβ1, TGFβ2, TGFβ3, bone morphogenetic protein (BMP)-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, and cartilage-derived morphogeneticprotein (CDMP); wherein said TGFβ superfamily proteins comprise fromabout 0.1% to about 50% of said mixture of proteins; ii. bone matrixproteins: osteocalcin, osteonectin, bone sialoprotein (BSP),lysyloxidase, and cathepsin L pre; wherein said bone matrix proteinscomprise from about 20% to about 98% of said mixture of proteins; and,iii. fibroblast growth factor-I (FGF-I), wherein said FGF-I comprisesfrom about 0.01% to about 50% of said mixture of proteins.
 2. Theproduct of claim 1, wherein said cartilage repair matrix isbioresorbable.
 3. The product of claim 1, wherein said cartilage repairmatrix is porous.
 4. The product of claim 1, wherein said cartilagerepair matrix comprises a material selected from the group consisting ofa synthetic polymeric material and a ground substance.
 5. The product ofclaim 4, wherein said ground substance is selected from the groupconsisting of natural polymers and proteoglycans.
 6. The product ofclaim 5, wherein said natural polymers are selected from the groupconsisting of collagen, elastin, and reticulin.
 7. The product of claim1, wherein said cartilage repair matrix contains from about 20% to about100% collagen by dry weight of said matrix.
 8. The product of claim 1,wherein said cartilage repair matrix contains from about 50% to about100% collagen by dry weight of said matrix.
 9. The product of claim 1,wherein said cartilage repair matrix contains from about 75% to about100% collagen by dry weight of said matrix.
 10. The product of claim 1,wherein said cartilage repair matrix comprises a collagen-containingmaterial selected from the group consisting of an autograft tissue, anallograft tissue, and a xenograft tissue.
 11. The product of claim 1,wherein said cartilage repair matrix comprises collagen from bovinetendon.
 12. The product of claim 1, wherein said matrix is configured asa tapered shape, and wherein said matrix is cross-linked.
 13. Theproduct of claim 12, wherein said cartilage repair matrix iscross-linked with an aldehyde.
 14. The product of claim 1, wherein saidmixture of proteins further comprises one or more serum proteins. 15.The product of claim 14, wherein said serum proteins are selected fromthe group consisting of albumin, transferrin, α2-Hs GlycoP, IgG,α1-antitrypsin, β2-microglobulin, Apo A1 lipoprotein (LP) and FactorXIIIb.
 16. The product of claim 14, wherein said serum proteins areselected from the group consisting of albumin, transferrin, Apo A1 LPand Factor XIIIb.
 17. The product of claim 1, wherein saidcartilage-inducing composition is at a concentration of from about 0.5%to about 33% by weight of said product.
 18. The product of claim 1,wherein said cartilage-inducing composition is at a concentration offrom about 1% to about 20% by weight of said product.
 19. The product ofclaim 1, wherein said mixture of proteins is bone protein (BP).
 20. Theproduct of claim 1, wherein said TGFβ superfamily proteins comprise fromabout 0.5% to about 25% of said mixture of proteins.
 21. The product ofclaim 1, wherein said TGFβ superfamily proteins comprise from about 1%to about 10% of said mixture of proteins.
 22. The product of claim 1,wherein said bone matrix proteins comprise from about 40% to about 98%of said mixture of proteins.
 23. The product of claim 1, wherein saidbone matrix proteins comprise from about 80% to about 98% of saidmixture of proteins.
 24. The product of claim 1, wherein said FGF-Icomprises from about 0.1% to about 10% of said mixture of proteins. 25.The product of claim 1, wherein said lesion is within meniscal cartilageselected from the group consisting of vascular meniscal cartilage andavascular meniscal cartilage.
 26. The product of claim 1, wherein saidcartilage inducing composition has an identifying characteristicselected from the group consisting of an ability to induce cellularinfiltration, an ability to induce cellular proliferation, and anability to induce cellular differentiation to type II collagen-producingchondrocytes.
 27. The product of claim 1, wherein saidcartilage-inducing composition further comprises a glycosaminoglycanthat non-covalently attaches to said proteins in said composition. 28.The product of claim 1, wherein said cartilage repair matrix comprises amaterial selected from the group consisting of poly(lactic acid),poly(glycolic acid), type I collagen, type II collagen, type IV collagenand hyaluronic acid.
 29. The product of claim 1, wherein said matrix isconfigured as a sheet, and wherein said sheet has a thickness of fromabout 0.1 mm to about 3 mm.
 30. The product of claim 1, wherein saidmatrix is configured as a sheet, and wherein said sheet has a thicknessof from about 0.5 mm to about 2 mm.
 31. The product of claim 1, whereinsaid matrix is configured as a sheet, and wherein said sheet is preparedfrom an aqueous dispersion of from about 0.2% to about 4% collagen byweight.
 32. The product of claim 1, wherein said matrix is configured asa sheet, and wherein said sheet is prepared from an aqueous dispersionof from about 0.5% to about 3% collagen by weight.
 33. The product ofclaim 1, wherein said cartilage repair matrix is configured as a sheet,and wherein said sheet is porous, having a pore diameter of from about10 μm to about 100 μm.
 34. The product of claim 1, wherein said matrixis configured as a tapered shape which varies in thickness from about0.5 mm to about 3 mm at its thinnest region to from about 4 mm to about10 mm at its thickest region.
 35. The product of claim 1, wherein saidmatrix is configured as a tapered shape, and wherein said matrix has adensity of from about 0.07 to about 0.5 grams matrix per cm³.
 36. Theproduct of claim 1, wherein said matrix is configured as a taperedshape, and wherein said matrix has a density of from about 0.1 to about0.25 grams matrix per cm³.
 37. The product of claim 1, wherein saidmatrix is configured as a tapered shape, and wherein said cartilagerepair matrix is porous, having a pore diameter of from about 50 μm toabout 500 μm.
 38. The product of claim 1, wherein said matrix isconfigured as a tapered shape, and wherein said cartilage repair matrixcomprises a porous ground substance composite which includes collagen.39. The product of claim 12, wherein said matrix is cross-linked by anagent selected from the group consisting of formaldehyde,glutaraldehyde, dimethyl suberimidate, carbodiimides, multi-functionalepoxides, succinimidyls, poly(glycidyl ether), diisocyanates, acylazide, tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysineand photo-oxidizers.
 40. The product of claim 12, wherein said matrix iscross-linked by a method selected from the group consisting of:ultraviolet irradiation and dehydrothermal treatment.
 41. The product ofclaim 1, wherein said cartilage-inducing composition is contained on orwithin said cartilage repair matrix by a method selected from the groupconsisting of freeze-drying said composition onto a surface of saidmatrix and suspension within said cartilage repair matrix of a deliveryformulation containing said composition.
 42. The product of claim 1,wherein said FGF-I comprises from about 0.05% to about 25% of saidmixture of proteins.
 43. A method for repair of meniscal cartilagelesions, comprising implanting and fixing into a meniscal cartilagelesion a product comprising: a. a cartilage repair matrix; and b. acartilage-inducing composition contained on or within said matrixcomprising a mixture of proteins comprising: i. transforming growthfactor β (TGFβ) superfamily proteins: TGFβ1, TGFβ2, TGFβ3, bonemorphogenetic protein (BMP)-2, BMP-3, BMP4, BMP-5, BMP-6, BMP-7, andcartilage-derived morphogenetic protein (CDMP); wherein said TGFβsuperfamily proteins comprise from about 0.1% to about 50% of saidmixture of proteins; ii. bone matrix proteins: osteocalcin, osteonectin,bone sialoprotein (BSP), lysyloxidase, and cathepsin L pre; wherein saidbone matrix proteins comprise from about 20% to about 98% of saidmixture of proteins; and, iii. fibroblast growth factor-I (FGF-I),wherein said FGF-I comprises from about 0.01% to about 50% of saidmixture of proteins.
 44. The method of claim 43, wherein said lesion isa tear and wherein said matrix is configured as a sheet, wherein saidstep of implanting comprises inserting said product directly into saidtear.
 45. The method of claim 43, wherein said product is fixed intosaid lesion by an attachment means selected from the group consisting ofbioresorbable sutures, non-resorbable sutures, press-fitting, arrows,nails, and T-fix suture anchor devices.
 46. The method of claim 43,wherein said lesion is a vascular meniscus lesion.
 47. The method ofclaim 43, wherein said lesion is an avascular meniscus lesion.
 48. Themethod of claim 43, wherein said lesion is a meniscal radial tear. 49.The method of claim 43, wherein said lesion is a meniscal bucket handletear.
 50. The method of claim 43, wherein said lesion is a meniscalsegmental defect.
 51. A product for repair of vascular and avascularmeniscus tears, comprising: a. a cartilage repair matrix comprisingcollagen selected from the group consisting of: a matrix configured as asheet which conforms to a meniscal radial tear, a matrix configured as asheet which conforms to a meniscal bucket handle tear, a matrixconfigured as a sheet which conforms to a meniscal longitudinal tear;and b. a cartilage-inducing composition contained on or within saidmatrix comprising a mixture of proteins comprising: i. transforminggrowth factor β (TGFβ) superfamily proteins: TGFβ1, TGFβ2, TGFβ3, bonemorphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, andcartilage-derived morphogenetic protein (CDMP); wherein said TGFβsuperfamily proteins comprise from about 0.1% to about 50% of saidmixture of proteins; ii. bone matrix proteins: osteocalcin, osteonectin,bone sialoprotein (BSP), lysyloxidase, and cathepsin L pre; wherein saidbone matrix proteins comprise from about 20% to about 98% of saidmixture of proteins; and, iii. fibroblast growth factor-I (FGF-I),wherein said FGF-I comprises from about 0.01% to about 50% of saidmixture of proteins.
 52. The product of claim 51, wherein said sheet isof a shape and size complementary to said tear.
 53. The product of claim51, wherein said tear is an avascular tear and said composition furthercomprises a time controlled delivery formulation.
 54. A method forrepair of vascular meniscus lesions, comprising implanting and fixinginto a cartilage lesion in the vascular region of a meniscus a productcomprising: a. a cartilage repair matrix comprising collagen andconfigured as a sheet; and b. a cartilage-inducing composition containedon or within said matrix comprising a mixture of proteins comprising: i.transforming growth factor β (TGFβ) superfamily proteins: TGFβ1, TGFβ2,TGFβ3, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6,BMP-7, and cartilage-derived morphogenetic protein (CDMP); wherein saidTGFβ superfamily proteins comprise from about 0.1% to about 50% of saidmixture of proteins; ii. bone matrix proteins: osteocalcin, osteonectin,bone sialoprotein (BSP), lysyloxidase, and cathepsin L pre; wherein saidbone matrix proteins comprise from about 20% to about 98% of saidmixture of proteins; and, iii. fibroblast growth factor-I (FGF-I),wherein said FGF-I comprises from about 0.01% to about 50% of saidmixture of proteins.
 55. The method of claim 54, wherein the rate ofrepair of said vascular mensicus lesion is measurably enhanced ascompared to the rate of repair of a meniscus lesion repaired in theabsence of said product.
 56. The method of claim 54, wherein the qualityof repair of said vascular mensicus lesion is measurably enhanced ascompared to the quality of repair of a meniscus lesion repaired in theabsence of said product.
 57. A product for repair of meniscal cartilagelesions, comprising: a. a cartilage repair matrix selected from thegroup consisting of: a matrix configured as a sheet which conforms to ameniscal radial tear, a matrix configured as a sheet which conforms to ameniscal bucket handle tear, a matrix configured as a sheet whichconforms to a meniscal longitudinal tear, a matrix configured as atapered shape which conforms to a segmental defect in a meniscus, and amatrix configured as a meniscus or a segmental portion thereof; and b. acartilage-inducing composition associated with said matrix, saidcomposition comprising at least BMP-3, BMP-2 and TGFβ1, wherein thequantity of BMP-3 in said composition is about 2-6 fold greater than thequantity of BMP-2 and about 10-30 fold greater than the quantity ofTGFP1 in said composition.
 58. The product of claim 57, wherein saidcomposition further comprises: (i) bone matrix proteins: osteocalcin,osteonectin, bone sialoprotein (BSP), lysyloxidase, and cathepsin L pre;wherein said bone matrix proteins comprise from about 20% to about 98%of said composition; and, (ii) fibroblast growth factor-I (FGF-I),wherein said FGF-I comprises from about 0.01% to about 50% of saidcomposition.